Prodrugs of 3-(pyrrol-2-ylmethylidene)-2-indolinone derivatives

ABSTRACT

The present invention is directed to prodrugs of certain 3-(pyrrol-2-yl-methylidene)-2-indolinone derivatives that modulate the activity of protein kinases (“PKs”). Pharmaceutical compositions comprising these compounds, methods of treating diseases related to abnormal PK activity utilizing pharmaceutical compositions comprising these compounds and methods of preparing them are also disclosed.

CROSS-REFERENCE

This application is a continuation of U.S. application Ser. No.10/243,942, filed Sep. 16, 2002, now U.S. Pat. No. 6,716,870, which is adivisional of U.S. application Ser. No. 09/863,819, filed May 24, 2001now U.S. Pat. No. 6,482,848, which claims priority under 35 U.S.C.119(e) to U.S. Provisional application Ser. Nos. 60/207,000 filed on May24, 2000, and 60/225,045 filed on Aug. 11, 2000, the disclosures ofwhich are incorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

1. Field of Invention

The present invention is directed to prodrugs of certain3-(pyrrol-2-ylmethylidene)-2-indolinone derivatives that modulate theactivity of protein kinases (“PKs”). Pharmaceutical compositionscomprising these compounds, methods of treating diseases related toabnormal PK activity utilizing pharmaceutical compositions comprisingthese compounds and methods of preparing them are also disclosed.

2. State of the Art

Protein kinases (“PKs”) are enzymes that catalyze the phosphorylation ofhydroxy groups on tyrosine, serine and threonine residues of proteins.PKs can be conveniently broken down into two classes, the proteintyrosine kinases (PTKs) and the serine-threonine kinases (STKs). One ofthe prime aspects of PTK activity is their involvement with growthfactor receptors. Growth factor receptors are cell-surface proteins.When bound by a growth factor ligand, growth factor receptors areconverted to an active form which interacts with proteins on the innersurface of a cell membrane. This leads to phosphorylation on tyrosineresidues of the receptor and other proteins and to the formation insidethe cell of complexes with a variety of cytoplasmic signaling moleculesthat, in turn, effect numerous cellular responses such as cell division(proliferation), cell differentiation, cell growth, expression ofmetabolic effects to the extracellular microenvironment, etc (See.,Schlessinger and Ullrich (1992) Neuron 9:303–391).

Growth factor receptors with PTK activity are known as receptor tyrosinekinases (“RTKs”). They comprise a large family of transmembranereceptors with diverse biological activity. At present, at leastnineteen (19) distinct subfamilies of RTKs have been identified. Anexample of these is the subfamily designated the “HER” RTKs, whichinclude EGFR (epithelial growth factor receptor), HER2, HER3 and HER4.

Another RTK subfamily consists of insulin receptor (IR), insulin-likegrowth factor I receptor (IGF-1R) and insulin receptor related receptor(IRR). IR and IGF-1R interact with insulin, IGF-I and IGF-II to form aheterotetramer of two entirely extracellular glycosylated α subunits andtwo β subunits which cross the cell membrane and which contain thetyrosine kinase domain.

A third RTK subfamily is referred to as the platelet derived growthfactor receptor (“PDGFR”) group, which includes PDGFRα, PDGFRβ, CSFIR,c-kit and c-fms. Another group is the fetus liver kinase (“flk”)receptor subfamily. This group is believed to be made of up of kinaseinsert domain-receptor fetal liver kinase-1 (KDR/FLK-1), flk-1R, flk-4and fms-like tyrosine kinase 1 (flt-1).

A further member of the tyrosine kinase growth factor receptor family isthe fibroblast growth factor (“FGF”) receptor subgroup. This groupconsists of four receptors, FGFR1–4, and seven ligands, FGF1–7. Whilenot yet well defined, it appears that the receptors consist of aglycosylated extracellular domain containing a variable number ofimmunoglobin-like loops and an intracellular domain in which thetyrosine kinase sequence is interrupted by regions of unrelated aminoacid sequences.

Still another member of the tyrosine kinase growth factor receptorfamily is the vascular endothelial growth factor (“VEGF”) receptorsubgroup. VEGF is a dimeric glycoprotein similar to PDGF but hasdifferent biological functions and target cell specificity in vivo. Inparticular, VEGF is presently thought to play an essential role isvasculogenesis and angiogenesis.

A more complete listing of the known RTK subfamilies is described inPlowman et al., DN&P, 7(6):334–339 (1994) which is incorporated byreference, including any drawings, as if fully set forth herein.

In addition to the RTKs, there also exists a family of entirelyintracellular PTKs called “non-receptor tyrosine kinases” or “cellulartyrosine kinases.” This latter designation, abbreviated “CTK,” will beused herein. CTKs do not contain extracellular and transmembranedomains. At present, over 24 CTKs in 11 subfamilies (Src, Frk, Btk, Csk,Abl, Zap70, Fes, Fps, Fak, Jak and Ack) have been identified. The Srcsubfamily appear so far to be the largest group of CTKs and includesSrc, Yes, Fyn, Lyn, Lck, Blk, Hck, Fgr and Yrk. For a more detaileddiscussion of CTKs, see Bolen, Oncogene, 8:2025–2031 (1993), which isincorporated by reference, including any drawings, as if fully set forthherein.

The serine/threonine kinases, STKs, like the CTKs, are predominantlyintracellular although there are a few receptor kinases of the STK type.STKs are the most common of the cytosolic kinases; i.e., kinases thatperform their function in that part of the cytoplasm other than thecytoplasmic organelles and cytoskelton. The cytosol is the region withinthe cell where much of the cell's intermediary metabolic andbiosynthetic activity occurs; e.g., it is in the cytosol that proteinsare synthesized on ribosomes.

RTKs, CTKs and STKs have all been implicated in a host of pathogenicconditions including, significantly, cancer. Other pathogenic conditionswhich have been associated with PTKs include, without limitation,psoriasis, hepatic cirrhosis, diabetes, angiogenesis, restenosis, oculardiseases, rheumatoid arthritis and other inflammatory disorders,immunological disorders such as autoimmune disease, cardiovasculardisease such as atherosclerosis and a variety of renal disorders.

With regard to cancer, two of the major hypotheses advanced to explainthe excessive cellular proliferation that drives tumor developmentrelate to functions known to be PK regulated. That is, it has beensuggested that malignant cell growth results from a breakdown in themechanisms that control cell division and/or differentiation. It hasbeen shown that the protein products of a number of proto-oncogenes areinvolved in the signal transduction pathways that regulate cell growthand differentiation. These protein products of proto-oncogenes includethe extracellular growth factors, transmembrane growth factor PTKreceptors (RTKs), cytoplasmic PTKs (CTKs) and cytosolic STKs, discussedabove.

In view of the apparent link between PK-related cellular activities andwide variety of human disorders, it is no surprise that a great deal ofeffort is being expended in an attempt to identify ways to modulate PKactivity. For example, attempts have been made to identify smallmolecules which act as PK inhibitors. For example, bis-monocylic,bicyclic and heterocyclic aryl compounds (PCT WO 92/20642),vinylene-azaindole derivatives (PCT WO 94/14808) and1-cyclopropyl-4-pyridylquinolones (U.S. Pat. No. 5,330,992) have beendescribed as tyrosine kinase inhibitors. Styryl compounds (U.S. Pat. No.5,217,999), styryl-substituted pyridyl compounds (U.S. Pat. No.5,302,606), quinazoline derivatives (EP Application No. 0 566 266 A1),selenaindoles and selenides (PCT WO 94/03427), tricyclic polyhydroxyliccompounds (PCT WO 92/21660), and benzylphosphonic acid compounds (PCT WO91/15495). Additionally, a family of novel pyrrole-substituted2-indolinone compounds have been discovered which exhibit PK modulatingability and have a salutary effect against disorders related to abnormalPK activity (U.S. Pat. No. 5,792,783 and PCT Application Publication No.WO 99/61422). Administration of various species of pyrrole-substituted2-indolinone compounds has been shown to be an effective therapeuticapproach to cure many kinds of solid tumors. For example,3-(3,5-dimethyl-1H-pyrrol-2-ylmethylidene)-1,3-dihydro-indol-2-one, ahighly active selective inhibitor of the vascular endothelial growthfactor receptor (Flk-1/KDR), inhibits tyrosine kinase catalysis, tumorvascularization, and growth of multiple tumor types (Fong et al. (1999)Cancer Res. 59:99–106). These compounds, however, have highlipophilicity and low solubility in water and most common vehicles atphysiological pH limit their adminstration.

Accordingly, there is a need for PK inhibitors that do not exhibit suchdrawbacks. The present invention fulfills this and related needs.

SUMMARY OF THE INVENTION

In one aspect, this invention relates to compounds having the formula I,II, III, or IV:

wherein:

R² is hydrogen;

R³, R⁴, R⁵ and R⁶ are independently selected from the group consistingof hydrogen, alkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl,heteroaryl, heteroalicyclic, hydroxy, alkoxy, aryloxy, mercapto,alkylthio, arylthio, sulfinyl, sulfonyl, S-sulfonamido, N-sulfonamido,trihalomethane-sulfonamido, carbonyl, C-carboxy, O-carboxy, C-amido,N-amido, cyano, nitro, halo, O-carbamyl, N-carbamyl, O-thiocarbamyl,N-thiocarbamyl, amino and —NR¹¹R¹² where R¹¹ and R¹² are independentlyselected from the group consisting of hydrogen, alkyl, cycloalkyl, aryl,carbonyl, acetyl, sulfonyl, and trifluoromethanesulfonyl, or R¹¹ andR¹², together with the nitrogen atom to which they are attached, combineto form a five- or six-member heteroalicyclic ring provided that atleast two of R³, R⁴, R⁵ and R⁶ are hydrogen; or

R³ and R⁴, R⁴ and R⁵, or R⁵ and R⁶ combine to form a six-membered arylring, a methylenedioxy or an ethylenedioxy group;

R⁷is selected from the group consisting of hydrogen, alkyl, cycloalkyl,alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy,aryloxy, carbonyl, acetyl, C-amido, C-thioamido, amidino, C-carboxy,O-carboxy, sulfonyl and trihalomethane-sulfonyl;

R⁸, R⁹ and R¹⁰ are independently selected from the group consisting ofhydrogen, alkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl,heteroaryl, heteroalicyclic, hydroxy, alkoxy, aryloxy, mercapto,alkylthio, arylthio, sulfinyl, sulfonyl, S-sulfonamido, N-sulfonamido,carbonyl, C-carboxy, O-carboxy, cyano, nitro, halo, O-carbamyl,N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, amino,-(alk₁)Z (where alk₁ is selected from the group consisting of alkyl,alkenyl or alkynyl and Z is hydroxy, alkoxy, carboxy, nitro, cyano,amino, guanidino, amido, ureido, sulfonamido, sulfinyl, sulfonyl,phosphonate, morpholino, piperazinyl or tetrazolyl) and —NR¹¹R¹² whereinR¹¹ and R¹² are as defined above;

R^(1′) is hydrogen or alkyl;

R^(2′) is hydrogen, alkyl, aralkyl, acyl, or —P(O)(OR)(OR′);

R^(3′) and R^(4′) are independently alkyl, or R^(3′) and R^(4′),together with the nitrogen atom to which they are attached, combine toform a heteroalicyclic ring or a heteroaryl ring provided that theheteroalicyclic ring is not piperidin-1-yl or morpholin-4-yl;

R^(5′) is alkyl;

R and R′ are independently selected from the group consisting ofhydrogen, alkyl, aralkyl and aryl; and

R^(a) and R^(b) are independently selected from hydrogen or alkyl; or apharmaceutically acceptable salt thereof.

Preferably, the invention is directed to a compound having the formulaI, III, or IV:

wherein:

R²is hydrogen;

R³, R⁴, R⁵ and R⁶ are independently selected from the group consistingof hydrogen, alkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl,heteroaryl, heteroalicyclic, hydroxy, alkoxy, aryloxy, mercapto,alkylthio, arylthio, sulfinyl, sulfonyl, S-sulfonamido, N-sulfonamido,trihalomethane-sulfonamido, carbonyl, C-carboxy, O-carboxy, C-amido,N-amido, cyano, nitro, halo, O-carbamyl, N-carbamyl, O-thiocarbamyl,N-thiocarbamyl, amino and —NR¹¹R¹² where R¹¹ and R¹² are independentlyselected from the group consisting of hydrogen, alkyl, cycloalkyl, aryl,carbonyl, acetyl, sulfonyl, and trifluoromethanesulfonyl, or R¹¹ andR¹², together with the nitrogen atom to which they are attached, combineto form a five- or six-membered heteroalicyclic ring provided that atleast two of R³, R⁴, R⁵ and R⁶ are hydrogen; or

R³ and R⁴, R⁴ and R⁵, or R⁵ and R⁶ combine to form a six-membered arylring, a methylenedioxy or an ethylenedioxy group;

R⁷ is selected from the group consisting of hydrogen, alkyl, cycloalkyl,alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy,aryloxy, carbonyl, acetyl, C-amido, C-thioamido, amidino, C-carboxy,O-carboxy, sulfonyl and trihalomethane-sulfonyl;

R⁸, R⁹ and R¹⁰ are independently selected from the group consisting ofhydrogen, alkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl,heteroaryl, heteroalicyclic, hydroxy, alkoxy, aryloxy, mercapto,alkylthio, arylthio, sulfinyl, sulfonyl, S-sulfonamido, N-sulfonamido,carbonyl, C-carboxy, O-carboxy, cyano, nitro, halo, O-carbamyl,N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, amino,-(alk₁)Z (where alk₁ is selected from the group consisting of alkyl,alkenyl or alkynyl and Z is hydroxy, alkoxy, carboxy, nitro, cyano,amino, guanidino, amido, ureido, sulfonamido, sulfinyl, sulfonyl,phosphonate, morpholino, piperazinyl or tetrazolyl) and —NR¹¹R¹² whereinR¹¹ and R¹² are as defined above;

R^(1′) is hydrogen or alkyl;

R^(2′) is hydrogen, alkyl, aralkyl, acyl, or —P(O)(OR)(OR′);

R^(5′) is alkyl;

R and R′ are independently selected from the group consisting ofhydrogen, alkyl, aralkyl and aryl;

R^(a) and R^(b) are independently selected from hydrogen or alkyl; or apharmaceutically acceptable salt thereof.

Specifically, the compounds of the present invention convert in vivo tocompounds of Formula V:

that exhibit PK modulating ability, in particular PK inhibiting ability,and are therefore useful in treating disorders related to abnormal PKactivity. The active compound (V) formed from the compounds of thepresent invention are described in U.S. Pat. No. 5,792,783, PCTApplication Publication No. WO 99/61422, and U.S. patent applicationSer. No. 09/783,264, filed on Feb. 15, 2001, and titled “PYRROLESUBSTITUTED 2-INDOLINONE AS PROTEIN KINASE INHIBITORS”, the disclosuresof which are hereby incorporated by reference. The prodrug compounds ofthe present invention have advantages over compounds of Formula (V) byvirtue of unexpected increased aqueous solubility over the parentcompound thus making them particularly suitable for intravenous (IV)formulations. A general description of the advantages and uses ofprodrugs as pharmaceutically useful compounds is given in an article byWaller and George in Br. J. Clin. Pharmac., Vol. 28, pp. 497–507, 1989.

In a second aspect this invention is directed to a pharmaceuticalcomposition comprising one or more compound(s) of Formula I–IV,preferably, I, III, or IV or a pharmaceutically acceptable salt thereofand a pharmaceutically acceptable excipient.

In a third aspect, this invention is directed to a method of treatingdiseases mediated by abnormal protein kinase activity, in particular,receptor tyrosine kinases (RTKs), non-receptor protein tyrosine kinases(CTKs) and serine/threonine protein kinases (STKs), in an organism, inparticular humans, which method comprises administering to said organisma pharmaceutical composition comprising a compound of Formula FormulaI–IV, preferably, I, III, or IV, or a pharmaceutically acceptable saltthereof and a pharmaceutically acceptable excipient. Such diseasesinclude by way of example and not limitation, cancer, diabetes, hepaticcirrhosis, cardiovascular disease such as atherosclerosis, angiogenesis,immunological disease such as autoimmune disease (e.g., AIDS and lupus)and renal disease. Specifically, the diseases mediated by EGF, HER2,HER3, HER4, IR, IGF-1R, IRR, PDGFRα, PDGFRβ, CSFIR, C-Kit, C-fms,Flk-1R, Flk4, KDR/Flk-1, Flt-1, FGFR-1R, FGFR-2R, FGFR-3R, FGFR-4R, Src,Frk, Btk, Csk, Abl, ZAP70, Fes/Fps, Fak, Jak, Ack, Yes, Fyn, Lyn, Lck,Blk, Hck, Fgr, Yrk, CDK2 and Raf.

In a fourth aspect, this invention is directed to a method of modulatingthe catalytic activity (e.g. inhibiting the catalytic activity) of PKs,in particular receptor tyrosine kinases (RTKs), non-receptor proteintyrosine kinases (CTKs) and serine/threonine protein kinases (STKs),using a compound of this invention or a pharmaceutical compositioncomprising a compound of this invention and a pharmaceuticallyacceptable excipient. The method may be carried out in vitro or in vivo.In particular, the receptor protein kinase whose catalytic activity ismodulated by a compound of this invention is selected from the groupconsisting of EGF, HER2, HER3, HER4, IR, IGF-1R, IRR, PDGFRα, PDGFRβ,CSFIR, C-Kit, C-fms, Flk-1R, Flk4, KDR/Flk-1, Flt-1, FGFR-1R, FGFR-2R,FGFR-3R and FGFR-4R. The cellular tyrosine kinase whose catalyticactivity is modulated by a compound of this invention is selected fromthe group consisting of Src, Frk, Btk, Csk, Abl, ZAP70, Fes/Fps, Fak,Jak, Ack, Yes, Fyn, Lyn, Lck, Blk, Hck, Fgr and Yrk. Theserine-threonine protein kinase whose catalytic activity is modulated bya compound of this invention is selected from the group consisting ofCDK2 and Raf.

In a fifth aspect, this invention is directed to the use of a compoundof formula Formula I–IV, preferably, I, III, or IV in the preparation ofa medicament useful in the treatment of a disease mediated by abnormalPK activity.

In a sixth aspect, this invention is directed to a method of preparing acompound of formula II which method comprises reacting a compound ofFormula V

with an amine of formula —NR^(3′)R^(4′) in the presence of an aldehydeof formula R^(1′)CHO where R^(1′), R^(3′) and R^(4′) are as defined informula II above;

optionally modifying any of the R³—R¹⁰ groups; and

optionally preparing an acid addition salt thereof.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Unless otherwise stated the following terms used in the specificationand claims have the meanings discussed below:

“Alkyl” refers to a saturated aliphatic hydrocarbon including straightchain, or branched chain groups. Preferably, the alkyl group has 1 to 20carbon atoms (whenever a numerical range; e.g., “1–20”, is statedherein, it means that the group, in this case the alkyl group, maycontain 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc. up to andincluding 20 carbon atoms). More preferably, it is a medium size alkylhaving 1 to 10 carbon atoms. Most preferably, it is a lower alkyl having1 to 4 carbon atoms e.g., methyl, ethyl, n-propyl, isopropyl, butyl,iso-butyl, tert-butyl and the like. The alkyl group may be substitutedor unsubstituted. When substituted, the substituent group(s) ispreferably one or more, more preferably one or two groups, individuallyselected from the group consisting of cycloalkyl, aryl, heteroaryl,heteroalicyclic, hydroxy, alkoxy, aryloxy, mercapto, alkylthio,arylthio, cyano, halo, carbonyl, thiocarbonyl, O-carbamyl, N-carbamyl,O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, C-carboxy, O-carboxy,nitro, silyl, amino, ammonium and —NR¹³R¹⁴ where R¹³ and R¹⁴ areindependently selected from the group consisting of hydrogen,unsubstituted alkyl, alkyl, cycloalkyl, aryl, carbonyl, acetyl,sulfonyl, amino, and trifluoromethanesulfonyl, or R¹³ and R¹⁴, togetherwith the nitrogen atom to which they are attached, combine to form afive- or six-member heteroalicyclic ring. More preferably, thesubstituent is hydroxy, amino, or —NR¹³R¹⁴ where R¹³ and R¹⁴ areindependently selected from the group consisting of unsubstituted alkyl,alkyl substituted with amino or hydroxy, or R¹³ and R¹⁴ together withthe nitrogen atom to which they are attached combine to formpyrrolidine, morpholine, or piperazine.

A “cycloalkyl” group refers to an all-carbon monocyclic ring (i.e.,rings which share an adjacent pair of carbon atoms) of 3 to 6 ring atomswherein one of more of the rings does not have a completely conjugatedpi-electron system e.g, cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, and the like.Examples, without limitation, of cycloalkyl groups are cyclopropane,cyclobutane, cyclopentane, cyclopentene, cyclohexane, adamantane,cyclohexadiene, cycloheptane and, cycloheptatriene. A cycloalkyl groupmay be substituted or unsubstituted. When substituted, the substituentgroup(s) is preferably one or more, more preferably one or two groups,individually selected from unsubstituted alkyl, alkyl, aryl, heteroaryl,unsubstituted heteroalicyclic, heteroalicyclic, hydroxy, alkoxy,aryloxy, mercapto, alkylthio, arylthio, cyano, halo, carbonyl,thiocarbonyl, C-carboxy, O-carboxy, O-carbamyl, N-carbamyl, C-amido,N-amido, nitro, amino and —NR¹³R¹⁴, with R¹³ and R¹⁴ as defined above.

An “alkenyl” group refers to an alkyl group, as defined herein,consisting of at least two carbon atoms and at least one carbon-carbondouble bond e.g., ethenyl, propenyl, butenyl or pentenyl and theirstructural isomeric forms such as 1- or 2-propenyl, 1-, 2-, or 3-butenyland the like.

An “alkynyl” group refers to an alkyl group, as defined herein,consisting of at least two carbon atoms and at least one carbon-carbontriple bond e.g., acetylene, ethnyl, propynyl, butynyl, or pentnyl andtheir structural isomeric forms as described above.

An “aryl” group refers to an all-carbon monocyclic or fused-ringpolycyclic (i.e., rings which share adjacent pairs of carbon atoms)groups of 6 to 12 ring atoms and having a completely conjugatedpi-electron system. Examples, without limitation, of aryl groups arephenyl, naphthalenyl and anthracenyl. The aryl group may be substitutedor unsubstituted. When substituted, the substituted group(s) ispreferably one or more, more preferably one, two, or three substituents,independently selected from the group consisting of halo, trihalomethyl, alkyl, hydroxy, alkoxy, aryloxy, mercapto, alkylthio, arylthio,cyano, nitro, carbonyl, thiocarbonyl, C-carboxy, O-carboxy, O-carbamyl,N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, sulfinyl,sulfonyl, amino and —NR¹³R¹⁴, with R¹³ and R¹⁴ as defined above.Preferably the substituent(s) is/are independently selected from chloro,fluoro, bromo, methyl, ethyl, propyl including all its isomeric forms,butyl including all its isomeric forms, hydroxy, methoxy, phenoxy, thio,methylthio, phenylthio, cyano, nitro, carboxy, methoxycarbonyl, oramino.

A “heteroaryl” group refers to a monocyclic or fused aromatic ring(i.e., rings which share an adjacent pair of atoms) of 5 to 9 ring atomsin which one, two, three or four ring atoms are selected from the groupconsisting of nitrogen, oxygen and sulfur and the rest being carbon.Examples, without limitation, of heteroaryl groups are pyrrole, furan,thiophene, imidazole, oxazole, thiazole, pyrazole, tetrazole, pyridine,pyrimidine, quinoline, isoquinoline, purine and carbazole. Theheteroaryl group may be substituted or unsubstituted. When substituted,the substituted group(s) is preferably one or more, more preferably oneor two substituents, independently selected from the group consisting ofalkyl, cycloalkyl, halo, trihalomethyl, hydroxy, alkoxy, aryloxy,mercapto, alkylthio, arylthio, cyano, nitro, carbonyl, thiocarbonyl,sulfonamido, C-carboxy, O-carboxy, sulfinyl, sulfonyl, O-carbamyl,N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, amino and—NR¹³R¹⁴, with R¹³ and R¹⁴ as defined above. Preferably thesubstituent(s) is/are independently selected from chloro, fluoro, bromo,methyl, ethyl, propyl including all its isomeric forms, butyl includingall its isomeric forms, hydroxy, methoxy, phenoxy, thio, methylthio,phenylthio, cyano, nitro, carboxy, methoxycarbonyl, or amino.

A “heteroalicyclic” group refers to a monocyclic or fused ring of 4 to 9ring atoms containing one, two, or three heteroatoms in the ring whichare selected from the group consisting of nitrogen, oxygen and —S(O)_(n)where n is 0–2, the remaining ring atoms being carbon. The rings mayalso have one or more double bonds. However, the rings do not have acompletely conjugated pi-electron system. Examples, without limitation,of heteroalicyclic groups are pyrrolidine, piperidine, piperazine,morpholine, imidazolidine, tetrahydropyridazine, tetrahydrofuran,thiomorpholine, tetrahydropyridine, and the like. The heteroalicyclicring may be substituted or unsubstituted. When substituted, thesubstituted group(s) is preferably one or more, more preferably one,two, or three substituents, independently selected from the groupconsisting of alkyl, cycloalkyl, halo, trihalomethyl, hydroxy, alkoxy,aryloxy, mercapto, alkylthio, arylthio, cyano, nitro, carbonyl,thiocarbonyl, C-carboxy, O-carboxy, O-carbamyl, N-carbamyl,O-thiocarbamyl, N-thiocarbamyl, sulfinyl, sulfonyl, C-amido, N-amido,amino and —NR¹³R¹⁴, with R¹³ and R¹⁴ as defined above. Preferably thesubstituent(s) is/are independently selected from chloro, fluoro, bromo,methyl, ethyl, propyl including all its isomeric forms, butyl includingall its isomeric forms, hydroxy, methoxy, phenoxy, thio, methylthio,phenylthio, cyano, nitro, carboxy, methoxycarbonyl, or amino.

A “hydroxy” group refers to an —OH group.

An “alkoxy” group refers to an —O-unsubstituted alkyl, —O-substitutedalkyl and an —O-unsubstitutedcycloalkyl group, as defined herein.Examples include and are not limited to methoxy, ethoxy, propoxy,butoxy, cyclopropyloxy, and the like, preferably methoxy.

An “aryloxy” group refers to both an —O-aryl and an —O-heteroaryl group,as defined herein. Examples include and are not limited to phenoxy,napthyloxy, pyridyloxy, furanyloxy, and the like.

A “mercapto” group refers to an —SH group.

A “alkylthio” group refers to both an S-alkyl and an —S-cycloalkylgroup, as defined herein. Examples include and are not limited tomethylthio, ethylthio, and the like.

A “arylthio” group refers to both an —S-aryl and an —S-heteroaryl group,as defined herein. Examples include and are not limited to phenylthio,napthylthio, pyridylthio, furanylthio, and the like.

A “sulfinyl” group refers to a —S(═O)—R″ group wherein, in addition tobeing as defined below, R″ may also be a hydroxy group, e.g.,methylsulfinyl, phenylsulfinyl, and the like.

A “sulfonyl” group refers to a —S(═O)₂R″ group wherein, in addition tobeing as defined below, R″ may also be a hydroxy group e.g.,methylsulfonyl, ethylsulfonyl, phenylsulfonyl, and the like.

A “trihalomethyl” group refers to a —CX₃ group wherein X is a halo groupas defined herein e.g., trifluoromethyl, trichloromethyl,tribromomethyl, dichlorofluoromethyl, and the like.

A “trihalomethanesulfonyl” group refers to a X₃CS(═O)₂— groups with X asdefined above, e.g., trifluoromethylsulfonyl, trichloromethylsulfonyl,tribromomethylsulfonyl, and the like.

A “trihalomethanesulfonylamido” group refers to a —NH—S(═O)₂R groupswherein R is trihalomethyl as defined above.

“Carbonyl” and “acyl” are used interchangeably herein to refer to a—C(═O)—R″ group, where R″ is selected from the group consisting ofhydrogen, alkyl, cycloalkyl, aryl, heteroaryl (bonded through a ringcarbon) and heteroalicyclic (bonded through a ring carbon), as definedherein. Representative examples include and are not limited to acetyl,propionyl, benzoyl, formyl, cyclopropylcarbonyl, pyridinylcarbonyl,pyrrolidin-1-ylcarbonyl, and the like

An “aldehyde” group refers to a carbonyl group where R″ is hydrogen.

A “thiocarbonyl” group refers to a —C(═S)—R″ group, with R″ as definedherein.

A “C-carboxy” group refers to a —C(═O)O—R″ group, with R″ as definedherein e.g., —COOH, methylcarbonyl, ethylcarbonyl, benzyloxycarbonyl,and the like.

An “O-carboxy” group refers to a —OC(═O)R″ group, with R″ as definedherein e.g., methylcarbonyloxy, phenylcarbonyloxy, benzylcarbonyloxy,and the like.

An “ester” group refers to a —C(═O)O—R″ group with R″ as defined hereinexcept that R″ cannot be hydrogen e.g., methoxycarbonyl,benzyloxycarbonyl, and the like.

An “acetyl” group refers to a —C(═O)CH₃ group.

A “carboxylic acid” group refers to a C-carboxy group in which R″ ishydrogen.

A “halo” group refers to fluorine, chlorine, bromine or iodine.

A “cyano” group refers to a —C≡N group.

A “nitro” group refers to a —NO₂ group.

A “methylenedioxy” group refers to a —OCH₂O— group where the two oxygenatoms are bonded to adjacent carbon atoms.

An “ethylenedioxy” group refers to a —OCH₂CH₂O— where the two oxygenatoms are bonded to adjacent carbon atoms.

An “S-sulfonamido” group refers to a —S(═O)₂NR¹³R¹⁴ group, with R¹³ andR¹⁴ as defined herein. Representative examples include and are notlimited to dimethylaminosulfonyl, aminosulfonyl,phenylmethylaminosulfonyl, phenylaminosulfonyl, and the like.

An “N-sulfonamido” group refers to a —NR¹³S(═O)₂R¹⁴ group, with R¹³ andR¹⁴ as defined herein e.g., methylsulfonylamino, ethylsulfonylamino,phenylsulfonylamino, benzylsulfonylamino, and the like.

An “O-carbamyl” group refers to a —OC(═O)NR¹³R¹⁴ group with R¹³ and R¹⁴as defined herein.

An “N-carbamyl” group refers to a R¹⁴OC(═O)NR¹³— group, with R¹³ and R¹⁴as defined herein.

An “O-thiocarbamyl” group refers to a —OC(═S)NR¹³R¹⁴ group with R¹³ andR¹⁴ as defined herein.

An “N-thiocarbamyl” group refers to a R¹⁴OC(═S)NR¹³— group, with R¹³ andR¹⁴ as defined herein.

An “amino” group refers to an —NR¹³R¹⁴ group, wherein R¹³ and R¹⁴ areindependently hydrogen or unsubstituted alkyl e.g, —NH₂, dimethylamino,diethylamino, ethylamino, methylamino, phenylamino, and the like.

A “C-amido” group refers to a —C(═O)NR¹³R¹⁴ group with R¹³ and R¹⁴ asdefined herein. Preferably R¹³ is hydrogen or unsubstituted lower alkyland R¹⁴ is hydrogen, lower alkyl optionally substituted withheteroalicyclic, hydroxy, or amino. For example, —C(═O)NR¹³R¹⁴ may beaminocarbonyl, dimethylaminocarbonyl, diethylaminocarbonyl,diethylaminoethylaminocarbonyl, ethylaminoethylaminocarbonyl,2-morpholinoethylaminocarbonyl, 3-morpholinopropylaminocarbonyl,3-morpholino-2-hydroxypropylaminocarbonyl, and the like.

An “N-amido” group refers to a R¹⁴C(═O)NR¹³— group, with R¹³ and R¹⁴ asdefined herein e.g., acetylamino, and the like.

A “ammonium” group refers to a —⁺NR¹⁵R¹⁶R¹⁷ group, wherein R¹⁵ and R¹⁶are independently selected from the group consisting of alkyl,cycloalkyl, aryl, and heteroaryl, and R¹⁷ is selected from the groupconsisting of hydrogen, alkyl, cycloalkyl, aryl, and heteroaryl.

A “amidino” group refers to a R¹⁵R¹⁶NC(═NR¹⁷)— group, with R¹⁵, R¹⁶ andR¹⁷ as defined above.

A “ureido” group refers to a —NR¹⁷C(═O)NR¹⁵R¹⁶ group, with R¹⁵, R¹⁶ andR¹⁷ as defined above.

A “guanidino” group refers to a —R¹³NC(═NR¹⁴)NR¹⁵R¹⁶ group, with R¹³,R¹⁴, R¹⁵ and R¹⁶ as defined above.

A “phosphonate” group refers to a —OP(═O)(OR)(OR′), with R and R′ asdefined herein.

A “tetrazolo” group refers to a group having the chemical structure:

A “morpholino” group refers to a group having the chemical structure

A “piperazinyl” group refers to a group having the chemical structure:

The terms “indolinone”, “2-indolinone” and “indolin-2-one” are usedinterchangeably herein to refer to a molecule having the chemicalstructure:

“Pyrrole” refers to a molecule having the chemical structure:

“Pyrrole-substituted 2-indolinone” and “3-pyrrol-1-yl-2-indolinone” areused interchangeably herein to refer to a chemical compound having thegeneral structure shown below:

A “prodrug” refers to an agent which is converted into the parent drugin vivo. Prodrugs are often useful because, in some situations, they maybe easier to administer than the parent drug. They may, for instance, bebioavailable by oral administration whereas the parent drug is not. Theprodrug may also have improved solubility in pharmaceutical compositionsover the parent drug. A prodrug may be converted into the parent drug byvarious mechanisms, including enzymatic processes and metabolichydrolysis. See Harper, “Drug Latentiation” in Jucker, ed. Progress inDrug Research 4:221–294 (1962); Morozowich et al., “Application ofPhysical Organic Principles to Prodrug Design” in E. B. Roche ed. Designof Biopharmaceutical Properties through Prodrugs and Analogs, APHA Acad.Pharm. Sci. (1977); Bioreversible Carriers in Drug in Drug Design,Theory and Application, E. B. Roche, ed., APHA Acad. Pharm. Sci. (1987);Design of Prodrugs, H. Bundgaard, Elsevier (1985); Wang et al. “Prodrugapproaches to the improved delivery of peptide drug” in Curr. Pharm.Design. 5(4):265–287 (1999); Pauletti et al. (1997) Improvement inpeptide bioavailability: Peptidomimetics and Prodrug Strategies, Adv.Drug. Delivery Rev. 27:235–256; Mizen et al. (1998) “The Use of Estersas Prodrugs for Oral Delivery of β-Lactam antibiotics,” Pharm. Biotech.11,:345–365; Gaignault et al. (1996) “Designing Prodrugs andBioprecursors I. Carrier Prodrugs,” Pract. Med. Chem. 671–696;Asgharnejad, “Improving Oral Drug Transport”, in Transport Processes inPharmaceutical Systems, G. L. Amidon, P. I. Lee and E. M. Topp, Eds.,Marcell Dekker, p. 185–218 (2000); Balant et al., “Prodrugs for theimprovement of drug absorption via different routes of administration”,Eur. J. Drug Metab. Pharmacokinet., 15(2):143–53 (1990); Balimane andSinko, “Involvement of multiple transporters in the oral absorption ofnucleoside analogues”, Adv. Drug Delivery Rev., 39(1–3): 183–209 (1999);Browne, “Fosphenytoin (Cerebyx)”, Clin. Neuropharmacol. 20(1): 1–12(1997); Bundgaard, “Bioreversible derivatization of drugs—principle andapplicability to improve the therapeutic effects of drugs”, Arch. Pharm.Chemi 86(1): 1–39 (1979); Bundgaard H. “Improved drug delivery by theprodrug approach”, Controlled Drug Delivery 17: 179–96 (1987); BundgaardH. “Prodrugs as a means to improve the delivery of peptide drugs”, Adv.Drug Delivery Rev. 8(1): 1–38 (1992); Fleisher et al. “Improved oraldrug delivery: solubility limitations overcome by the use of prodrugs”,Adv. Drug Delivery Rev. 19(2): 115–130 (1996); Fleisher et al. “Designof prodrugs for improved gastrointestinal absorption by intestinalenzyme targeting”, Methods Enzymol. 112 (Drug Enzyme Targeting, Pt. A):360–81, (1985); Farquhar D, et al., “Biologically ReversiblePhosphate-Protective Groups”, J. Pharm. Sci., 72(3): 324–325 (1983);Freeman S, et al., “Bioreversible Protection for the Phospho Group:Chemical Stability and Bioactivation of Di(4-acetoxy-benzyl)Methylphosphonate with Carboxyesterase,” J. Chem. Soc., Chem. Commun.,875–877 (1991); Friis and Bundgaard, “Prodrugs of phosphates andphosphonates: Novel lipophilic alpha-acyloxyalkyl ester derivatives ofphosphate- or phosphonate containing drugs masking the negative chargesof these groups”, Eur. J Pharm. Sci. 4: 49–59 (1996); Gangwar et al.,“Pro-drug, molecular structure and percutaneous delivery”, Des.Biopharm. Prop. Prodrugs Analogs, [Symp.] Meeting Date 1976, 409–21.(1977); Nathwani and Wood, “Penicillins: a current review of theirclinical pharmacology and therapeutic use”, Drugs 45(6): 866–94 (1993);Sinhababu and Thakker, “Prodrugs of anticancer agents”, Adv. DrugDelivery Rev. 19(2): 241–273 (1996); Stella et al., “Prodrugs. Do theyhave advantages in clinical practice?”, Drugs 29(5): 455–73 (1985); Tanet al. “Development and optimization of anti-HIV nucleoside analogs andprodrugs: A review of their cellular pharmacology, structure-activityrelationships and pharmacokinetics”, Adv. Drug Delivery Rev. 39(1–3):117–151 (1999); Taylor, “Improved passive oral drug delivery viaprodrugs”, Adv. Drug Delivery Rev., 19(2): 131–148 (1996); Valentino andBorchardt, “Prodrug strategies to enhance the intestinal absorption ofpeptides”, Drug Discovery Today 2(4): 148–155 (1997); Wiebe and Knaùs,“Concepts for the design of anti-HIV nucleoside prodrugs for treatingcephalic HIV infection”, Adv. Drug Delivery Rev.: 39(1–3):63–80 (1999);Walter et al., “Prodrugs”, Br. J. Clin. Pharmac. 28:.497–507 (1989).

The compounds of this invention may possess one or more chiral centers,and can therefore be produced as individual stereoisomers or as mixturesof stereoisomers, depending on whether individual stereoisomers ormixtures of stereoisomers of the starting materials are used. Unlessindicated otherwise, the description or naming of a compound or group ofcompounds is intended to include both the individual stereoisomers ormixtures (racemic or otherwise) of stereoisomers. Methods for thedetermination of stereochemistry and the separation of stereoisomers arewell known to a person of ordinary skill in the art [see the discussionin Chapter 4 of March J: Advanced Organic Chemistry, 4th ed. John Wileyand Sons, New York, N.Y., 1992].

The chemical formulae referred to herein may exhibit the phenomena oftautomerism and structural isomerism. For example, the compoundsdescribed herein may adopt an E or a Z configuration about the doublebond connecting the 2-indolinone moiety to the pyrrole moiety or theymay be a mixture of E and Z. This invention encompasses any tautomericor structural isomeric form and mixtures thereof.

The term “method” refers to manners, means, techniques and proceduresfor accomplishing a given task including, but not limited to, thosemanners, means, techniques and procedures either known to, or readilydeveloped from known manners, means, techniques and procedures by,practitioners of the chemical, pharmaceutical, biological, biochemicaland medical arts.

As used herein, the term “modulation” or “modulating” refers to thealteration of the catalytic activity of RTKs, CTKs and STKs. Inparticular, modulating refers to the activation of the catalyticactivity of RTKs, CTKs and STKs, preferably the activation or inhibitionof the catalytic activity of RTKs, CTKs and STKs, depending on theconcentration of the compound or salt to which the RTK, CTK or STK isexposed or, more preferably, the inhibition of the catalytic activity ofRTKs, CTKs and STKs.

The term “catalytic activity” as used herein refers to the rate ofphosphorylation of tyrosine under the influence, direct or indirect, ofRTKs and/or CTKs or the phosphorylation of serine and threonine underthe influence, direct or indirect, of STKs.

The term “contacting” as used herein refers to bringing a compound ofthis invention and a target PK together in such a manner that thecompound can affect the catalytic activity of the PK, either directly,i.e., by interacting with the kinase itself, or indirectly, i.e., byinteracting with another molecule on which the catalytic activity of thekinase is dependent. Such “contacting” can be accomplished in vitro,i.e., in a test tube, a petri dish or the like. In a test tube,contacting may involve only a compound and a PK of interest or it mayinvolve whole cells. Cells may also be maintained or grown in cellculture dishes and contacted with a compound in that environment. Inthis context, the ability of a particular compound to affect a PKrelated disorder, i.e., the IC₅₀ of the compound, defined below, can bedetermined before use of the compounds in vivo with more complex livingorganisms is attempted. For cells outside the organism, multiple methodsexist, and are well-known to those skilled in the art, to get the PKs incontact with the compounds including, but not limited to, direct cellmicroinjection and numerous transmembrane carrier techniques.

“In vitro” refers to procedures performed in an artificial environmentsuch as, e.g., without limitation, in a test tube or culture medium. Theskilled artisan will understand that, for example, an isolated PK may becontacted with a modulator in an in vitro environment. Alternatively, anisolated cell may be contacted with a modulator in an in vitroenvironment.

As used herein, “in vivo” refers to procedures performed within a livingorganism such as, without limitation, a mouse, rat, rabbit, ungulate,bovine, equine, porcine, canine, feline, primate, or human.

As used herein, “PK related disorder,” “PK driven disorder,” and“abnormal PK activity” all refer to a condition characterized byinappropriate, i.e., under or, more commonly, over PK catalyticactivity, where the particular PK can be an RTK, a CTK or an STK.Inappropriate catalytic activity can arise as the result of either: (1)PK expression in cells which normally do not express PKs, (2) increasedPK expression leading to unwanted cell proliferation, differentiationand/or growth, or, (3) decreased PK expression leading to unwantedreductions in cell proliferation, differentiation and/or growth.Over-activity of a PK refers to either amplification of the geneencoding a particular PK or production of a level of PK activity whichcan correlate with a cell proliferation, differentiation and/or growthdisorder (that is, as the level of the PK increases, the severity of oneor more of the symptoms of the cellular disorder increases).Under-activity is, of course, the converse, wherein the severity of oneor more symptoms of a cellular disorder increase as the level of the PKactivity decreases.

The term “organism” refers to any living entity comprised of at leastone cell. A living organism can be as simple as, for example, a singleeukaryotic cell or as complex as a mammal, including a human being.

The term “therapeutically effective amount” as used herein refers tothat amount of the compound being administered which will relieve tosome extent one or more of the symptoms of the disorder being treated.In reference to the treatment of cancer, a therapeutically effectiveamount refers to that amount which has the effect of (1) reducing thesize of the tumor, (2) inhibiting (that is, slowing to some extent,preferably stopping) tumor metastasis, (3) inhibiting to some extent(that is, slowing to some extent, preferably stopping) tumor growth,and/or, (4) relieving to some extent (or, preferably, eliminating) oneor more symptoms associated with the cancer.

“Pharmaceutically acceptable salt” refers to those salts which retainthe biological effectiveness and properties of the free bases and whichare obtained by reaction with inorganic or organic acids such ashydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, methanesulfonic acid, ethanesulfonic acid,p-toluenesulfonic acid, salicylic acid, malic acid, maleic acid,succinic acid, tartaric acid, citric acid, and the like.

A “pharmaceutical composition” refers to a mixture of one or more of thecompounds described herein, or a pharmaceutically acceptable saltsthereof, with other chemical components, such as physiologicallyacceptable carriers and excipients. The purpose of a pharmaceuticalcomposition is to facilitate administration of a compound to anorganism.

As used herein, a “pharmaceutically acceptable carrier” refers to acarrier or diluent that does not cause significant irritation to anorganism and does not abrogate the biological activity and properties ofthe administered compound.

An “excipient” refers to an inert substance added to a pharmaceuticalcomposition to further facilitate administration of a compound.Examples, without limitation, of excipients include calcium carbonate,calcium phosphate, various sugars and types of starch, cellulosederivatives, gelatin, vegetable oils and polyethylene glycols.

“Treating” or “treatment” of a disease includes preventing the diseasefrom occurring in an animal that may be predisposed to the disease butdoes not yet experience or exhibit symptoms of the disease (prophylactictreatment), inhibiting the disease (slowing or arresting itsdevelopment), providing relief from the symptoms or side-effects of thedisease (including palliative treatment), and relieving the disease(causing regression of the disease). With regard to cancer, these termssimply mean that the life expectancy of an individual affected with acancer will be increased or that one or more of the symptoms of thedisease will be reduced.

By “monitoring” is meant observing or detecting the effect of contactinga compound with a cell expressing a particular PK. The observed ordetected effect can be a change in cell phenotype, in the catalyticactivity of a PK or a change in the interaction of a PK with a naturalbinding partner. Techniques for observing or detecting such effects arewell-known in the art. For example, the catalytic activity of a PK maybe observed by determining the rate or amount of phosphorylation of atarget molecule. The above-referenced effect is selected from a changeor an absence of change in a cell phenotype, a change or absence ofchange in the catalytic activity of said protein kinase or a change orabsence of change in the interaction of said protein kinase with anatural binding partner in a final aspect of this invention.

“Cell phenotype” refers to the outward appearance of a cell or tissue orthe biological function of the cell or tissue. Examples, withoutlimitation, of a cell phenotype are cell size, cell growth, cellproliferation, cell differentiation, cell survival, apoptosis, andnutrient uptake and use. Such phenotypic characteristics are measurableby techniques well-known in the art.

A “natural binding partner” refers to a polypeptide that binds to aparticular PK in a cell. Natural binding partners can play a role inpropagating a signal in a PK-mediated signal transduction process. Achange in the interaction of the natural binding partner with the PK canmanifest itself as an increased or decreased concentration of thePK/natural binding partner complex and, as a result, in an observablechange in the ability of the PK to mediate signal transduction.

PRESENTLY PREFERRED EMBODIMENTS

While the broadest definition of the invention is set out in the Summaryof the Invention, certain compounds of this invention are presentlypreferred.

-   (A) A preferred group of compounds is represented by formula (I):

wherein:

R² is hydrogen;

R³, R⁴, R⁵, and R⁶ are independently selected from the group consistingof hydrogen, alkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl,heteroaryl, heteroalicyclic, hydroxy, alkoxy, aryloxy, mercapto,alkylthio, arylthio, sulfinyl, sulfonyl, S-sulfonamido, N-sulfonamido,trihalomethane-sulfonamido, carbonyl, C-carboxy, O-carboxy, C-amido,N-amido, cyano, nitro, halo, O-carbamyl, N-carbamyl, O-thiocarbamyl,N-thiocarbamyl, amino, and —NR¹¹R¹² where R¹¹ and R¹² are independentlyselected from the group consisting of hydrogen, alkyl, cycloalkyl, aryl,carbonyl, acetyl, sulfonyl, and trifluoromethanesulfonyl, or R¹¹ andR¹², together with the nitrogen atom to which they are attached, combineto form a five- or six-membered heteroalicyclic ring provided that atleast two of R³, R⁴, R⁵ and R⁶ are hydrogen; or

R³ and R⁴, R⁴ and R⁵, or R⁵ and R⁶ combine to form a six-membered arylring, a methylenedioxy or an ethylenedioxy group;

R⁷ is selected from the group consisting of hydrogen, alkyl, cycloalkyl,alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy,aryloxy, carbonyl, acetyl, C-amido, C-thioamido, amidino, C-carboxy,O-carboxy, sulfonyl and trihalomethane-sulfonyl;

R⁸, R⁹ and R¹⁰ are independently selected from the group consisting ofhydrogen, alkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl,heteroaryl, heteroalicyclic, hydroxy, alkoxy, aryloxy, mercapto,alkylthio, arylthio, sulfinyl, sulfonyl, S-sulfonamido, N-sulfonamido,carbonyl, C-carboxy, O-carboxy, cyano, nitro, halo, O-carbamyl,N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, amino,-(alk₁)Z (where alk₁ is selected from the group consisting of alkyl,alkenyl or alkynyl and Z is hydroxy, alkoxy, carboxy, nitro, cyano,amino, guanidino, amido, ureido, sulfonamido, sulfinyl, sulfonyl,phosphonate, morpholino, piperazinyl or tetrazolyl) and —NR¹¹R¹² whereinR¹¹ and R¹² are as defined above;

R^(1′) is hydrogen or alkyl;

R^(2′) is hydrogen, alkyl, aralkyl, acyl or —P(O)(OR)(OR′) where R andR′ are independently selected from the group consisting of hydrogen,alkyl, aralkyl or aryl; or a pharmaceutically acceptable salt thereof.

-   (a) In a preferred embodiment of formula I, R^(2′) and R⁷ are    hydrogen.

Within this group a more preferred group of compounds is that wherein:

-   -   R³ is hydrogen or lower unsubstituted alkyl, preferably hydrogen        or methyl, more preferably hydrogen;    -   R⁴ is selected from the group consisting of hydrogen, halogen,        aryl and S-sulfonamido, preferably hydrogen, chloro, fluoro,        bromo, phenyl, even more preferably hydrogen or fluoro, most        preferably hydrogen;    -   R⁵ is selected from the group consisting of hydrogen, lower        alkyl, lower alkoxy, aryl, and heteroaryl, preferably hydrogen,        methyl, ethyl, methoxy, phenyl, pyridyl, more preferably        hydrogen; and    -   R^(1′) and R⁶ hydrogen.

Within the above preferred and more preferred groups an even morepreferred group of compounds is that wherein:

-   -   R⁸ and R¹⁰ are unsubstituted lower alkyl, preferably methyl; and        R⁹ is hydrogen, C-amido, or -(alk₁)Z (where alk₁ is selected        from the group consisting of alkyl, alkenyl or alkynyl and Z is        hydroxy, alkoxy, carboxy, nitro, cyano, amino, guanidino, amido,        ureido, sulfonamido, sulfinyl, sulfonyl, phosphonate,        morpholino, piperazinyl or tetrazolyl), preferably hydrogen,        2-(dimethylaminoethyl)aminocarbonyl,        2-(diethylaminoethyl)aminocarbonyl,        2-(pyrrolidin-1-ylethyl)aminocarbonyl,        2-(morpholin-4-ylethyl)aminocarbonyl, or 3-carboxypropyl, more        preferably hydrogen.

Particularly preferred compounds within this group are:

(3Z)-3-[(3,5-dimethyl-1H-pyrrol-2-yl)-methylidene]-1-(hydroxymethyl)-1,3-dihydro-2H-indol-2-one(1) and3-[5-{(Z)-[1-(hydroxymethyl)-1,2-dihydro-3H-indol-3-ylidine]methyl}-2,4-dimethyl-1H-pyrrole-3-propanoicacid (5).

-   (b) Another preferred group of compounds of formula I is that    wherein:

R^(2′) is —P(O)(OR)(OR′) and R⁷ is hydrogen.

Within this group a more preferred group of compounds is that wherein:

-   -   R³ is hydrogen or lower unsubstituted alkyl, preferably hydrogen        or methyl, more preferably hydrogen;    -   R⁴ is selected from the group consisting of hydrogen, halogen,        aryl and S-sulfonamido, preferably hydrogen, chloro, fluoro,        bromo, phenyl, even more preferably hydrogen or fluoro, most        preferably hydrogen;    -   R⁵ is selected from the group consisting of hydrogen, lower        alkyl, lower alkoxy, aryl, and heteroaryl, preferably hydrogen,        methyl, ethyl, methoxy, phenyl, pyridyl, more preferably        hydrogen; and    -   R^(1′) and R⁶ are hydrogen.

Within the above preferred and more preferred groups an even morepreferred group of compounds is that wherein:

-   -   R⁸ and R¹⁰ are unsubstituted lower alkyl, preferably methyl; and        R⁹ is hydrogen, C-amido, or -(alk₁)Z (where alk₁ is selected        from the group consisting of alkyl, alkenyl or alkynyl and Z is        hydroxy, alkoxy, carboxy, nitro, cyano, amino, guanidino, amido,        ureido, sulfonamido, sulfinyl, sulfonyl, phosphonate,        morpholino, piperazinyl or tetrazolyl), preferably hydrogen,        2-(dimethylaminoethyl)aminocarbonyl,        2-(diethylaminoethyl)aminocarbonyl,        2-(pyrrolidin-1-ylethyl)aminocarbonyl,        2-(morpholin-4-ylethyl)aminocarbonyl, or 3-carboxypropyl, more        preferably hydrogen.

Within this group, particularly preferred compounds are{(3Z)-3-[(3,5-dimethyl-1H-pyrrol-2-yl)methylidene]-2-oxo-2,3-dihydro-1H-indol-1-yl}methyldihydrogen phosphate (7) and{(3Z)-3-[(3,5-dimethyl-4-(3-carboxypropyl)-1H-pyrrol-2-yl)methylidene]-2-oxo-2,3-dihydro-1H-indol-1-yl}methyldihydrogen phosphate.

-   (c) Yet another preferred group of compounds of formula I is that    wherein R^(2′) is acyl and R⁷ is hydrogen.

Within this group a more preferred group of compounds is that wherein:

-   -   R³ is hydrogen or lower unsubstituted alkyl, preferably hydrogen        or methyl, more preferably hydrogen;    -   R⁴ is selected from the group consisting of hydrogen, halogen,        aryl and S-sulfonamido, preferably hydrogen, chloro, fluoro,        bromo, phenyl, even more preferably hydrogen or fluoro, most        preferably hydrogen;    -   R⁵ is selected from the group consisting of hydrogen, lower        alkyl, lower alkoxy, aryl, and heteroaryl, preferably hydrogen,        methyl, ethyl, methoxy, phenyl, pyridyl, more preferably        hydrogen; and    -   R^(1′) and R⁶ are hydrogen.

Within the above preferred and more preferred groups an even morepreferred group of compounds is that wherein:

-   -   R⁸ and R¹⁰ are unsubstituted lower alkyl, preferably methyl; and        R⁹ is hydrogen, C-amido, or -(alk₁)Z (where alk₁ is selected        from the group consisting of alkyl, alkenyl or alkynyl and Z is        hydroxy, alkoxy, carboxy, nitro, cyano, amino, guanidino, amido,        ureido, sulfonamido, sulfinyl, sulfonyl, phosphonate,        morpholino, piperazinyl or tetrazolyl), preferably hydrogen,        2-(dimethylaminoethyl)aminocarbonyl,        2-(diethylaminoethyl)aminocarbonyl,        2-(pyrrolidin-1-ylethyl)aminocarbonyl,        2-(morpholin-4-ylethyl)aminocarbonyl, or 3-carboxypropyl, more        preferably hydrogen.

Within this group, particularly preferred compounds are4-({(3Z)-3-[(3,5-dimethyl-1H-pyrrol-2-yl)-methylidene]-2-oxo-2,3-dihydro-1H-indol-1-yl}methoxy)-4-oxobutanoicacid (3) and4-({(3Z)-3-[(3,5-dimethyl-4-(3-carboxypropyl)-1H-pyrrol-2-yl)-methylidene]-2-oxo-2,3-dihydro-1H-indol-1-yl}methoxy)-4-oxobutanoicacid.

-   (B) Another preferred group of compounds is represented by formula    (II):

-    wherein:

R² is hydrogen;

R³, R⁴, R⁵ and R⁶ are independently selected from the group consistingof hydrogen, alkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl,heteroaryl, heteroalicyclic, hydroxy, alkoxy, aryloxy, mercapto,alkylthio, arylthio, sulfinyl, sulfonyl, S-sulfonamido, N-sulfonamido,trihalomethane-sulfonamido, carbonyl, C-carboxy, O-carboxy, C-amido,N-amido, cyano, nitro, halo, O-carbamyl, N-carbamyl, O-thiocarbamyl,N-thiocarbamyl, amino and —NR¹¹R¹² where R¹¹ and R¹² are independentlyselected from the group consisting of hydrogen, alkyl, cycloalkyl, aryl,carbonyl, acetyl, sulfonyl, and trifluoromethanesulfonyl, or R¹¹ andR¹², together with the nitrogen atom to which they are attached, combineto form a five- or six-membered heteroalicyclic ring provided that atleast two of R³, R⁴, R⁵ and R⁶ are hydrogen; or

R³ and R⁴, R⁴ and R⁵, or R⁵ and R⁶ combine to form a six-membered arylring, a methylenedioxy or an ethylenedioxy group;

R⁷ is selected from the group consisting of hydrogen, alkyl, cycloalkyl,alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy,aryloxy, carbonyl, acetyl, C-amido, C-thioamido, amidino, C-carboxy,O-carboxy, sulfonyl and trihalomethane-sulfonyl;

R⁸, R⁹ and R¹⁰ are independently selected from the group consisting ofhydrogen, alkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl,heteroaryl, heteroalicyclic, hydroxy, alkoxy, aryloxy, mercapto,alkylthio, arylthio, sulfinyl, sulfonyl, S-sulfonamido, N-sulfonamido,carbonyl, C-carboxy, O-carboxy, cyano, nitro, halo, O-carbamyl,N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, amino,-(alk₁)Z (where alk₁ is selected from the group consisting of alkyl,alkenyl or alkynyl and Z is hydroxy, alkoxy, carboxy, nitro, cyano,amino, guanidino, amido, ureido, sulfonamido, sulfinyl, sulfonyl,phosphonate, morpholino, piperazinyl or tetrazolyl) and —NR¹¹R¹² whereinR¹¹ and R¹² are as defined above;

R^(1′) is hydrogen or alkyl; and

R^(3′) and R^(4′) are independently alkyl or together with the nitrogenatom to which they are attached combine to form a heteroalicyclic ringor a heteroaryl ring; or a pharmaceutically acceptable salt thereof;with the proviso that R^(3′) and R^(4′) are not morpholin-1-yl orpiperidin-1-yl.

Presently preferred compounds of formula II are:

-   -   R³ is hydrogen or lower unsubstituted alkyl, preferably hydrogen        or methyl, more preferably hydrogen;    -   R⁴ is selected from the group consisting of hydrogen, halogen,        aryl and S-sulfonamido, preferably hydrogen, chloro, fluoro,        bromo, phenyl, even more preferably hydrogen or fluoro, most        preferably hydrogen;    -   R⁵ is selected from the group consisting of hydrogen, lower        alkyl, lower alkoxy, aryl, and heteroaryl, preferably hydrogen,        methyl, ethyl, methoxy, phenyl, pyridyl, more preferably        hydrogen; and    -   R⁶ is hydrogen;    -   R⁷ is hydrogen;    -   R^(1′) is hydrogen or methyl, especially hydrogen;    -   R⁸ and R¹⁰ are independently unsubstituted lower alkyl,        especially methyl;    -   R⁹ is hydrogen, lower alkyl substituted with C-carboxy,        —C(═O)NHR¹² wherein R¹² is lower alkyl substituted with amino or        heteroalicyclic and optionally substituted with hydroxy; R⁹ is        preferably hydrogen, 3-carboxypropyl,        (2-diethylaminoethyl)-aminocarbonyl,        (2-ethylaminoethyl)aminocarbonyl,        2-(pyrrolidin-1-ylethyl)-aminocarbonyl,        3-(morpholin-4-yl)propyl-aminocarbonyl,        3-(morpholin-4-yl)-2-hydroxypropylaminocarbonyl; R⁹ is most        preferably hydrogen, 3-carboxypropyl,        (2-diethylaminoethyl)aminocarbonyl, or        (2-ethylaminoethyl)-aminocarbonyl; and    -   R^(3′) and R^(4′) are lower alkyl optionally substituted        hydroxy, especially methyl, ethyl, 2-hydroxyethyl; or R^(3′) and        R^(4′), together with the nitrogen atom to which they are        attached, form pyrrolidin-1-yl,        2-(S)-hydroxymethylpyrrolidin-1-yl,        2-(S)-carboxy-pyrrolidin-1-yl, piperazin-1-yl, or        4-methylpiperazin-1-yl, especially pyrrolidin-1-yl; or    -   R^(3′) and R^(4′), together with the nitrogen atom to which they        are attached form a heteroaryl ring, preferably, pyrro-1-yl,        pyridin-1-yl, oxazol-3-yl, isoxazol-2-yl, pyrazin-1-yl,        pyradizin-1-yl, quinolin-1-yl, imidazol-1-yl, more preferably        pyridin-1-yl.

A number of different preferences have been given above, and followingany one of these preferences results in a compound of this inventionthat is more presently preferred than a compound in which thatparticular preference is not followed. However, these preferences aregenerally independent [although some (alternative) preferences aremutually exclusive], and additive; and following more than one of thesepreferences may result in a more presently preferred compound than onein which fewer of the preferences are followed.

Presently preferred classes of compounds of formula II include thosewhere:

(a) R^(1′), R³, R⁴, R⁵, R⁶, R⁷, and R⁹ are hydrogen; R⁸ and R¹⁰ areunsubstituted lower alkyl, especially methyl; and R^(3′) and R^(4′),together with the nitrogen atom to which they are attached, formpyrrolidin-1-yl, 2-(S)-hydroxymethylpyrrolidin-1-yl,2-(S)-carboxypyrrolidin-1-yl, piperazin-1-yl, or 4-methylpiperazin-1-yl,especially pyrrolidin-1-yl.

(b) R^(1′), R³, R⁴, R⁵, R⁶, and R⁷ are hydrogen; R⁸ and R¹⁰ areunsubstituted lower alkyl, especially methyl; R⁹ is lower alkylsubstituted with C-carboxy, especially 3-carboxypropyl; and R^(3′) andR^(4′), together with the nitrogen atom to which they are attached, formpyrrolidin-1-yl, 2-(S)-hydroxymethylpyrrolidin-1-yl,2-(S)-carboxy-pyrrolidin-1-yl, piperazin-1-yl, or4-methylpiperazin-1-yl, especially pyrrolidin-1-yl.

(c) R^(1′), R³, R⁵, R⁶, and R⁷ are hydrogen; R⁴ is halo, especiallyfluoro, R⁸ and R¹⁰ are unsubstituted lower alkyl, especially methyl; R⁹is —C(═O)NHR¹³ wherein R¹³ is lower alkyl substituted with amino orheteroalicyclic and optionally substituted with hydroxy, especially(2-diethylaminoethyl)-aminocarbonyl, (2-ethylaminoethyl)-aminocarbonyl,2-(pyrrolidin-1-ylethyl)aminocarbonyl,3-(morpholin-4-yl)propylaminocarbonyl,3-(morpholin-4-yl)-2-hydroxypropylaminocarbonyl, particularly(2-diethylaminoethyl)aminocarbonyl, or(2-ethylaminoethyl)-aminocarbonyl; and R^(3′) and R^(4′), together withthe nitrogen atom to which they are attached, form pyrrolidin-1-yl,2-(S)-hydroxymethylpyrrolidin-1-yl, 2-(S)-carboxypyrrolidin-1-yl,piperazin-1-yl, or 4-methylpiperazin-1-yl, especially pyrrolidin-1-yl.

(d) R^(1′), R³, R⁴, R⁵, R⁶, R⁷, and R⁹ are hydrogen; R⁸ and R¹⁰ areunsubstituted lower alkyl, especially methyl; and R^(3′) and R^(4′),together with the nitrogen atom to which they are attached, form aheteroaryl ring, preferably, pyrrol-1-yl, pyridin-1-yl, oxazol-3-yl,isoxazol-2-yl, pyrazin-1-yl, pyridazin-1-yl, quinolin-1-yl,imidazol-1-yl, more preferably pyridin-1-yl.

Presently preferred compounds of formula II include:

(3Z)-3-[(3,5-dimethyl-1H-pyrrol-2-yl)-methylidene]-1-(1-pyrrolidinylmethyl)-1,3-dihydro-2H-indol-2-one(14);(3Z)-3-[(3,5-dimethyl-1H-pyrrol-2-yl)-methylidene]-1-(4-methylpiperazin-1-ylmethyl)-1,3-dihydro-2H-indol-2-one(13);(3Z)-3-[(3,5-dimethyl-1H-pyrrol-2-yl)-methylidene]-1-[2(S)-hydroxymethyl-1-pyrrolidinylmethyl)-1,3-dihydro-2H-indol-2-one;(3Z)-3-[(3,5-dimethyl-1H-pyrrol-2-yl)-methylidene]-1-[2(S)-carboxy-1-pyrrolidinylmethyl)-1,3-dihydro-2H-indol-2-one;(3Z)-3-{[3,5-dimethyl-4-(2-diethylaminoethylaminocarbonyl)-1H-pyrrol-2-yl]-methylidene}-1-(1-pyrrolidinylmethyl)-1,3-dihydro-2H-indol-2-one;(3Z)-3-{[3,5-dimethyl-4-(2-ethylaminoethylaminocarbonyl)-1H-pyrrol-2-yl]-methylidene}-1-(1-pyrrolidinylmethyl)-1,3-dihydro-2H-indol-2-one;and(3Z)-3-{[3,5-dimethyl-4-(3-morpholin-4-yl-2-hydroxypropylaminocarbonyl)-1H-pyrrol-2-yl]-methylidene}-1-(1-pyrrolidinylmethyl)-1,3-dihydro-2H-indol-2-one;and1-({(3Z)-3-[(3,5-dimethyl-1H-pyrrol-2-yl)-methylidene]-2-oxo-1,3-dihydro-1H-indol-1-yl}methyl)pyridiniumchloride (12).

-   (C) Another preferred group of compounds is represented by formula    III:

-    wherein:

R² is hydrogen;

R³, R⁴, R⁵ and R⁶ are independently selected from the group consistingof hydrogen, alkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl,heteroaryl, heteroalicyclic, hydroxy, alkoxy, aryloxy, mercapto,alkylthio, arylthio, sulfinyl, sulfonyl, S-sulfonamido, N-sulfonamido,trihalomethane-sulfonamido, carbonyl, C-carboxy, O-carboxy, C-amido,N-amido, cyano, nitro, halo, O-carbamyl, N-carbamyl, O-thiocarbamyl,N-thiocarbamyl, amino and —NR¹¹R¹² where R¹¹ and R¹² are independentlyselected from the group consisting of hydrogen, alkyl, cycloalkyl, aryl,carbonyl, acetyl, sulfonyl, and trifluoromethanesulfonyl, or R¹¹ andR¹², together with the nitrogen atom to which they are attached, combineto form a five- or six-membered heteroalicyclic ring provided that atleast two of R³, R⁴, R⁵ and R⁶ are hydrogen; or

R³ and R⁴, R⁴ and R⁵, or R⁵ and R⁶ combine to form a six-membered arylring, a methylenedioxy or an ethylenedioxy group;

R⁷ is selected from the group consisting of hydrogen, alkyl, cycloalkyl,alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy,aryloxy, carbonyl, acetyl, C-amido, C-thioamido, amidino, C-carboxy,O-carboxy, sulfonyl and trihalomethane-sulfonyl;

R⁸, R⁹ and R¹⁰ are independently selected from the group consisting ofhydrogen, alkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl,heteroaryl, heteroalicyclic, hydroxy, alkoxy, aryloxy, mercapto,alkylthio, arylthio, sulfinyl, sulfonyl, S-sulfonamido, N-sulfonamido,carbonyl, C-carboxy, O-carboxy, cyano, nitro, halo, O-carbamyl,N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, amino,-(alk₁)Z (where alk₁ is selected from the group consisting of alkyl,alkenyl or alkynyl and Z is hydroxy, alkoxy, carboxy, nitro, cyano,amino, guanidino, amido, ureido, sulfonamido, sulfinyl, sulfonyl,phosphonate, morpholino, piperazinyl or tetrazolyl) and —NR¹¹R¹² whereinR¹¹ and R¹² are as defined above; and

R^(5′) is alkyl; or

a pharmaceutically acceptable salt thereof.

Within this group a more preferred group of compounds is that wherein:

-   -   R³ is hydrogen or lower unsubstituted alkyl, preferably hydrogen        or methyl, more preferably hydrogen;    -   R⁴ is selected from the group consisting of hydrogen, halogen,        aryl and S-sulfonamido, preferably hydrogen, chloro, fluoro,        bromo, phenyl, even more preferably hydrogen or fluoro, most        preferably hydrogen;    -   R⁵ is selected from the group consisting of hydrogen, lower        alkyl, lower alkoxy, aryl, and heteroaryl, preferably hydrogen,        methyl, ethyl, methoxy, phenyl, pyridyl, more preferably        hydrogen; and    -   R⁶ and R⁷ are hydrogen.

Within the above preferred and more preferred groups an even morepreferred group of compounds is that wherein:

-   -   R⁸ and R¹⁰ are unsubstituted lower alkyl, preferably methyl; and        R⁹ is hydrogen, C-amido, or -(alk₁)Z (where alk₁ is selected        from the group consisting of alkyl, alkenyl or alkynyl and Z is        hydroxy, alkoxy, carboxy, nitro, cyano, amino, guanidino, amido,        ureido, sulfonamido, sulfinyl, sulfonyl, phosphonate,        morpholino, piperazinyl or tetrazolyl), preferably hydrogen,        2-(dimethylaminoethyl)aminocarbonyl,        2-(diethylaminoethyl)aminocarbonyl,        2-(pyrrolidin-1-ylethyl)aminocarbonyl,        2-(morpholin-4-ylethyl)aminocarbonyl, or 3-carboxypropyl, more        preferably hydrogen.

Within the above preferred group, a more preferred group of compoundsIII is that wherein R^(5′) is alkyl substituted with NR¹¹R¹² where R¹¹and R¹² are as defined above, or ammonium.

Particularly preferred compounds of formula III are2-{(3Z)-3-[(3,5-dimethyl-1H-pyrrol-2-yl)-methylidene]-2-oxo-2,3-dihydro-1H-indol-1-yl}-N,N,N-trimethyl-2-oxo-1-ethanaminiumchloride (18) and2-{(3Z)-3-[(3,5-dimethyl-4-(3-carboxypropyl)-1H-pyrrol-2-yl)-methylidene]-2-oxo-2,3-dihydro-1H-indol-1-yl}-N,N,N-trimethyl-2-oxo-1-ethanaminiumchloride.

-   (D) Yet another preferred group of compounds is represented by    formula IV:

-    wherein:

R² is hydrogen;

R³, R⁴, R⁵ and R⁶ are independently selected from the group consistingof hydrogen, alkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl,heteroaryl, heteroalicyclic, hydroxy, alkoxy, aryloxy, mercapto,alkylthio, arylthio, sulfinyl, sulfonyl, S-sulfonamido, N-sulfonamido,trihalomethane-sulfonamido, carbonyl, C-carboxy, O-carboxy, C-amido,N-amido, cyano, nitro, halo, O-carbamyl, N-carbamyl, O-thiocarbamyl,N-thiocarbamyl, amino and —NR¹¹R¹² where R¹¹ and R¹² are independentlyselected from the group consisting of hydrogen, alkyl, cycloalkyl, aryl,carbonyl, acetyl, sulfonyl, and trifluoromethanesulfonyl or R¹¹ and R¹²together with the nitrogen atom to which they are attached combine toform a five- or six-membered heteroalicyclic ring provided that at leasttwo of R³, R⁴, R⁵ and R⁶ are hydrogen; or

R³ and R⁴, R⁴ and R⁵, or R⁵ and R⁶ combine to form a six-membered arylring, a methylenedioxy or an ethylenedioxy group;

R⁷ is selected from the group consisting of hydrogen, alkyl, cycloalkyl,alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy,aryloxy, carbonyl, acetyl, C-amido, C-thioamido, amidino, C-carboxy,O-carboxy, sulfonyl and trihalomethane-sulfonyl;

R⁸, R⁹ and R¹⁰ are independently selected from the group consisting ofhydrogen, alkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl,heteroaryl, heteroalicyclic, hydroxy, alkoxy, aryloxy, mercapto,alkylthio, arylthio, sulfinyl, sulfonyl, S-sulfonamido, N-sulfonamido,carbonyl, C-carboxy, O-carboxy, cyano, nitro, halo, O-carbamyl,N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, amino,-(alk₁)Z (where alk₁ is selected from the group consisting of alkyl,alkenyl or alkynyl and Z is hydroxy, alkoxy, carboxy, nitro, cyano,amino, guanidino, amido, ureido, sulfonamido, sulfinyl, sulfonyl,phosphonate, morpholino, piperazinyl or tetrazolyl) and —NR¹¹R¹² whereinR¹¹ and R¹² are as defined above; and

R^(a) and R^(b) are independently selected from hydrogen or alkyl; or apharmaceutically acceptable salt thereof.

Within this group a more preferred group of compounds is that wherein:

-   -   R³ is hydrogen or lower unsubstituted alkyl, preferably hydrogen        or methyl, more preferably hydrogen;    -   R⁴ is selected from the group consisting of hydrogen, halogen,        aryl and S-sulfonamido, preferably hydrogen, chloro, fluoro,        bromo, phenyl, even more preferably hydrogen or fluoro, most        preferably hydrogen;    -   R⁵ is selected from the group consisting of hydrogen, lower        alkyl, lower alkoxy, aryl, and heteroaryl, preferably hydrogen,        methyl, ethyl, methoxy, phenyl, pyridyl, more preferably        hydrogen; and    -   R⁶ and R⁷ are hydrogen.

Within the above preferred and more preferred groups an even morepreferred group of compounds is that wherein:

-   -   R⁸ and R¹⁰ are unsubstituted lower alkyl, preferably methyl; and        R⁹ is hydrogen, C-amido, or -(alk₁)Z (where alk₁ is selected        from the group consisting of alkyl, alkenyl or alkynyl and Z is        hydroxy, alkoxy, carboxy, nitro, cyano, amino, guanidino, amido,        ureido, sulfonamido, sulfinyl, sulfonyl, phosphonate,        morpholino, piperazinyl or tetrazolyl), preferably hydrogen,        2-(dimethylaminoethyl)aminocarbonyl,        2-(diethylaminoethyl)aminocarbonyl,        2-(pyrrolidin-1-ylethyl)aminocarbonyl,        2-(morpholin-4-ylethyl)aminocarbonyl, or 3-carboxypropyl, more        preferably hydrogen.

Within the above preferred group, a more preferred group of compounds IVis that wherein R^(a) and R^(b) are independently hydrogen orunsubstituted lower alkyl, preferably hydrogen or methyl, mostpreferably hydrogen.

Particularly preferred compounds of formula IV are(3Z)-3-[(3,5-dimethyl-1H-pyrrol-2-yl)-methylidene]-1-(dimethylphosphonyl)-1,3-dihydro-2H-indol-2-one,(3Z)-3-[(3,5-dimethyl-1H-pyrrol-2-yl)-methylidene]-1-(phosphonyl)-1,3-dihydro-2H-indol-2-one,(3Z)-3-[(3,5-dimethyl-4-(3-carboxypropyl)-1H-pyrrol-2-yl)-methylidene]-1-(dimethylphosphonyl)-1,3-dihydro-2H-indol-2-one,and(3Z)-3-[(3,5-dimethyl-4-(3-carboxypropyl)-1H-pyrrol-2-yl)-methylidene]-1-(phosphonyl)-1,3-dihydro-2H-indol-2-one.

Other preferred group of compounds of this invention are those wherein:

R² is hydrogen.

R⁷ is hydrogen.

R³, R⁴, R⁵ and R⁶ are selected from the group consisting of hydrogen,unsubstituted lower alkyl, lower alkyl substituted with a group selectedfrom the group consisting of hydroxy, halo, C-carboxy substituted with agroup selected from the group consisting of hydrogen and unsubstitutedlower alkyl, amino, or —NR¹¹R¹²; unsubstituted lower alkyl alkoxy, loweralkoxy substituted with one or more halo groups, lower alkoxysubstituted with a group consisting of unsubstituted aryl or arylsubstituted with one or more groups independently selected from thegroup consisting of unsubstituted lower alkyl, hydroxy, unsubstitutedlower alkyl alkoxy, halo, amino, unsubstituted lower alkyl S-sulfonamidoor —NR¹¹R¹², unsubstituted aryl or aryl substituted with one or moregroups independently selected from the group consisting of unsubstitutedlower alkyl, unsubstituted lower alkyl alkoxy, lower alkoxy substitutedwith one or more halo groups, lower alkoxy substituted with a groupselected from the group consisting of unsubstituted aryl or arylsubstituted with one or more groups independently selected from thegroup consisting of unsubstituted lower alkyl, hydroxy, unsubstitutedlower alkyl alkoxy, halo, amino, unsubstituted lower alkyl S-sulfonamidoor —NR¹¹R¹², hydroxy, amino, unsubstituted lower alkyl sulfonamido,C-carboxy substituted with a groups selected from the group consistingof hydrogen or unsubstituted lower alkyl, morpholino, —NR¹¹R¹²,trihalomethyl, aryl, aryl substituted with one or more groupsindependently selected from the group consisting of hydroxy, halo,trihalomethyl, amino, —NR¹¹R¹², sulfonamido, C-carboxy substituted witha group selected from the group consisting of hydrogen or unsubstitutedlower alkyl, unsubstituted lower alkyl or lower alkyl substituted with agroup selected from the group consisting of hydroxy, halo, C-carboxysubstituted with a group selected from the group consisting of hydrogenor unsubstituted lower alkyl, amino or —NR¹¹R¹², unsubstitutedheteroalicyclic, heteroalicyclic substituted with one or more groupsindependently selected from the group consisting of halo, hydroxy,unsubstituted lower alkyl, unsubstituted lower alkyl carbonyl, hydroxy,unsubstituted lower alkyl alkoxy or alkoxy substituted with one or morehalo groups, unsubstituted aryloxy, aryloxy substituted with one or moregroups independently selected from the group consisting of unsubstitutedlower alkyl, trihalomethyl, halo, hydroxy, amino or —NR¹¹R¹², mercapto,unsubstituted lower alkyl alkylthio, unsubstituted arylthio, arylthiosubstituted with one or more groups selected from the group consistingof halo, hydroxy, amino or —NR¹¹R¹², C-carboxy substituted with a groupselected from the group consisting of hydrogen and unsubstituted loweralkyl, unsubstituted lower alkyl O-carboxy, unsubstituted lower alkylS-sulfonamido, nitro, unsubstituted lower alkyl C-amido, unsubstitutedlower alkyl N-amido, amino and —NR¹¹R¹².

More preferably, R³, R⁴, R⁵ and R⁶ are independently selected from thegroup consisting of hydrogen, halo, unsubstituted lower alkyl, loweralkyl substituted with one or more groups selected from the groupconsisting of hydroxy, halo, C-carboxy substituted with a group selectedfrom the group consisting of hydrogen or unsubstituted lower alkyl,amino or —NR¹¹R¹², unsubstituted lower alkyl alkoxy, lower alkyl alkoxysubstituted with one or more halo groups, unsubstituted aryloxy, aryloxysubstituted with one or more groups independently selected from thegroup consisting of unsubstituted lower alkyl, lower alkyl substitutedwith one or more halo groups, hydroxy unsubstituted lower alkyl alkoxy,halo, amino or —NR¹¹R¹², S-sulfonamido wherein R¹¹ and R¹² areindependently selected from the group consisting of hydrogen andunsubstituted lower alkyl, unsubstituted aryl, aryl substituted with oneor more groups independently selected from the group consisting of halo,unsubstituted lower alkyl, lower alkyl substituted with one or more halogroups, unsubstituted lower alkyl alkoxy, amino or —NR¹¹R¹²,unsubstituted heteroaryl, heteroaryl substituted with one or more groupsindependently selected from the group consisting of unsubstituted loweralkyl, lower alkyl substituted with one or more halo groups,unsubstituted lower alkyl alkoxy, hydroxy, halo, amino or —NR¹¹R¹²,unsubstituted heteroalicyclic, heteroalicyclic substituted with one ormore groups independently selected from the group consisting of halo,hydroxy, unsubstituted lower alkyl, lower alkyl substituted with one ormore halo groups, unsubstituted lower alkyl alkoxy, amino or —NR¹¹R¹²,unsubstituted lower alkyl O-carboxy, C-amido wherein R¹¹ and R¹² areindependently selected from the group consisting of hydrogen,unsubstituted lower alkyl and unsubstituted aryl, and, N-amido whereinR¹¹ and R¹² are independently selected from the group consisting ofhydrogen, unsubstituted lower alkyl and unsubstituted aryl. Even morepreferably, R³ R⁴, R⁵, and R⁶ are independently selected from the groupconsisting of hydrogen, halo, unsubstituted lower alkyl, lower alkylsubstituted with one or more hydroxy groups, unsubstituted lower alkoxy,unsubstituted aryl, aryl substituted with one or more unsubstitutedlower alkoxy groups, and —S(O)₂NR¹¹R¹², R⁵ is hydrogen, R⁶ is —NR¹¹R¹²,and R¹¹ and R¹² are independently selected from the group consisting ofhydrogen, unsubstituted lower alkyl and, R¹¹ and R¹², together with thenitrogen to which they are attached, combine to form a five-member or asix-member unsubstituted heteroalicyclic ring. Particularly preferably,R³, R⁴, R⁵ and R⁶ are all hydrogen or R³, R⁵ and R⁶ are hydrogen and R⁴is halo, preferably chloro, bromo or fluoro, more preferably fluoro.

R⁸, R⁹ and R¹⁰ may be -(alk₁)Z while the other two are independentlyselected from the group consisting of hydrogen, hydroxy, unsubstitutedlower alkyl, unsubstituted lower alkenyl, unsubstituted lower alkynyl,unsubstituted lower alkyl alkoxy, lower alkoxy substituted with one ormore halo groups, unsubstituted aryl alkoxy, amino, —NR¹¹R¹², halo,C-carboxy substituted with a groups selected from the group consistingof hydrogen or unsubstituted lower alkyl, unsubstituted lower alkylO-carboxy, unsubstituted lower alkyl C-amido, unsubstituted lower alkylN-amido, acetyl, unsubstituted lower alkyl S-sulfonamido, unsubstitutedaryl or aryl substituted with a group selected from the group consistingof halo, hydroxy, unsubstituted lower alkyl alkoxy, alkoxy substitutedwith one or more halo groups, C-carboxy substituted with a groupsselected from the group consisting of hydrogen or unsubstituted loweralkyl, unsubstituted lower alkyl O-carboxy, amino, unsubstituted loweralkyl S-sulfonamido and —NR¹¹R¹², preferably alk₁ is an unsubstitutedlower alkyl group (more preferably 2 to 4 carbon atoms) and Z isselected from the group consisting of hydroxy, amino, —NR¹¹R¹²,quaternary ammonium, C-carboxy substituted with a group selected fromthe group consisting of hydrogen or unsubstituted lower alkyl, C-amidosubstituted with groups selected from the group consisting of hydrogenand unsubstituted lower alkyl, morpholino, piperadinyl, tetrazolo andphosphonyl. Preferably, R⁸ and R¹⁰ are selected from the groupsconsisting of hydrogen and unsubstituted lower alkyl, R⁹ is selectedfrom the group consisting of hydrogen and alk₁Z.

It is likewise a presently preferred feature of this invention that R¹¹and R¹² are independently selected from the group comprising hydrogen,unsubstituted lower alkyl, hydroxy, unsubstituted lower alkyl alkoxy,unsubstituted lower alkyl carbonyl, unsubstituted lower alkyl O-carboxyand acetyl.

It is also a presently preferred embodiment of this invention that Z isselected from the group consisting of —C(═O)NR¹¹R¹² wherein R¹¹ and R¹²are independently selected from the group consisting of hydrogen,unsubstituted lower alkyl, lower alkyl substituted with a group selectedfrom the group consisting of amino and —NR¹¹R¹², unsubstituted aryl,aryl substituted with one or more groups selected from the groupconsisting of halo, hydroxy, unsubstituted lower alkyl alkoxy andtrihalomethyl, unsubstituted heteroaryl, unsubstituted heteroalicyclic,and R¹¹ and R¹², together with the nitrogen to which they are attached,combine to form a five-member or a six-member unsubstitutedheteroalicyclic, and, —NR¹¹R¹², wherein, R¹¹ and R¹² are independentlyselected from the group consisting of unsubstituted lower alkyl and,combined, a five-member or a six-member unsubstituted heteroalicyclicring.

General Synthetic Scheme

The starting materials and reagents used in preparing these compoundsare either available from commercial suppliers such as Aldrich ChemicalCo., (Milwaukee, Wis.), Bachem (Torrance, Calif.), or Sigma (St. Louis,Mo.) or are prepared by methods known to those skilled in the artfollowing procedures set forth in references such as Fieser and Fieser'sReagents for Organic Synthesis, Volumes 1–17 (John Wiley and Sons,1991); Rodd's Chemistry of Carbon Compounds, Volumes 1–5 andSupplementals (Elsevier Science Publishers, 1989); Organic Reactions,Volumes 1–40 (John Wiley and Sons, 1991), March's Advanced OrganicChemistry, (John Wiley and Sons, 4th Edition) and Larock's ComprehensiveOrganic Transformations (VCH Publishers Inc., 1989). These schemes aremerely illustrative of some methods by which the compounds of thisinvention can be synthesized, and various modifications to these schemescan be made and will be suggested to one skilled in the art havingreferred to this disclosure. The starting materials and theintermediates of the reaction may be isolated and purified if desiredusing conventional techniques, including but not limited tofiltration,distillation, crystallization, chromatography and the like. Suchmaterials may be characterized using conventional means, includingphysical constants and spectral data. Unless specified to the contrary,the reactions described herein take place at atmospheric pressure over atemperature range from about −78.degree ° C. to about 150° C., morepreferably from about 0° C. to about 125° C. and most preferably atabout room (or ambient) temperature, e.g., about 20° C.

Compounds of Formula I can be prepared as illustrated and describedbelow:

Compounds of Formula I where R^(2′) is hydrogen can be readily preparedfrom compounds of Formula V by condensing V with a suitable aldehyde offormula R^(′)CHO. The reaction may be carried out in the presence of anorganic base, preferably a tertiary nitrogen base such astrimethylamine, triethylamine, pyridine, diisopropylethylamine,1,8-diazabicyclo-[5.4.1]-undec-7-ene, and the like. The solvent in whichthe reaction is carried out may be an aprotic solvent. Examples, withoutlimitation, include pentane, hexane, benzene, toluene, methylenechloride, carbon tetrachloride, chloroform, tetrahydrofuran (THF),dimethylsulfoxide (DMSO), dimethylformamide (DMF), pyridine, and thelike. In a presently preferred embodiment of this invention, the solventis a polar aprotic protic solvent, preferably acetonitrile,dimethylformamide, tetrahydrofuran or pyridine. The reaction may becarried out at room temperature. Aldehydes of formula R^(1′)CHO arecommerically available or they can be prepared by methods well known inthe art. Some such examples, include but are not limited to,formaldehyde, acetaldehyde, proponaldehyde, an butyraldehyde arecommercially available.

A compound of Formula I where R^(2′) is hydrogen can be converted toother compounds of Formula I where R^(2′) is alkyl, aralkyl, aryl, acylor —P(O)(OR)(OR′) by methods well known in the art. Some such methodsare described below.

A compound of Formula I where R^(2′) is alkyl or aralkyl can be preparedby reacting I where R^(2′) is hydrogen with an alkylating agent of theformula R^(2′)X where R^(2′) is alkyl or aralkyl and X is a suitableleaving group such as halo, tosylate or mesylate, triflate, and thelike, in the presence of a base such as triethylamine, pyridine, and thelike. Alkylating agents such as methyl bromide, methyl iodide, benzylbromide, benzyl iodide, 2-phenylethyl chloride, ethyl bromide arecommercially available.

A compound of Formula I where R^(2′) is acyl can be prepared by reactingby reacting I where R^(2′) is hydrogen with an acylating agent such asacid anhydride e.g., acetic anhydride, succinic anhydride, and the like,acid halides such as acetyl chloride, propionyl chloride, butrylchloride an the like and carboxylic acid active esters such asp-nitrophenyl ester, pentafluorophenyl ester, and the like. The reactionis carried out in an organic base such as pyridine, DMAP, and the like.The reaction is carried out at ambient temperature. Alternatively,compounds of Formula I, where R^(2′) is acyl may be prepared by reactingthe parent 3-pyrrolidinyl-2-indolinone (V) with a suitable aldehyde suchas formaldehyde, acetaldehyde and the like, in the presence of asuitable acylating agent R²′X discussed above, without isolating theintermediate N-hydroxyalkyl derivative of V.

A compound of Formula I where R^(2′) is —P(O)(OR)(OR′) where R and R′are not hydrogen can be prepared by reacting by reacting I where R^(2′)is hydrogen with a phosphorylating agent in an organic base such astriethylamine, pyridine, and the like. Phosphorylating agent such asdibenzyl phosphorochloridate and benzyl methyl phosphorochloridate arecommercially available. A compound of Formula I where R and R′ arehydrogen can be prepared from a corresponding compound of Formula Iwhere R and R′ are benzyl by removal of the benzyl groups underhydrogenation reaction conditions. The reaction may be carried out inthe presence of a base. The base may be an organic or an inorganic base.If an organic base is used, preferably it is a tertiary nitrogen base,or an alkali metal alkoxide, e.g., sodium methoxide. Examples oftertiary nitrogen bases include, but are not limited to, trimethylamine,triethylamine, pyridine, and 1,8-diazabicyclo[5.4.1]undec-7-ene.Examples of inorganic bases are, without limitation, alkali metalhydrides such as sodium hydride and alkali metal hydroxides such assodium methoxide.

The solvent in which the reaction is carried out may be an aproticsolvent such as dimethylformamide, tetrahydrofuran or dimethylsulfoxide.

Compounds of Formula V can be prepared by methods well known in the art.For example, compound V where R³–R⁶, R⁷, and R⁹ are hydrogen and R⁸ andR¹⁰ are methyl can be prepared by following the procedure described inU.S. Pat. No. 5,792,783, at column 22, lines 60–67, the disclosure ofwhich is incorporated herein by reference. Other compounds of Formula(II) can be prepared as described in U.S. Pat. No. 5,792,783, PCTApplication Publication No. WO 99/61422, and U.S. patent applicationSer. No. 09/783,264, filed on Feb. 15, 2001, and titled “PYRROLESUBSTITUTED 2-INDOLINONE AS PROTEIN KINASE INHIBITORS”, the disclosuresof which are hereby incorporated by reference.

Compounds of Formula II where R^(3′) and R^(4′) are independently alkylor combine to form a heteroalicyclic ring may be prepared as illustratedand described below:

A compound of Formula I where R³–R¹⁰ and R^(1′), R^(3′) and R^(4′) areas described in the Summary of the Invention can be prepared by reactinga compound of formula V with an aldehyde such as formaldehyde,acetaldehyde, and the like, and a suitable amine.

The solvent in which the reaction is carried out may be a protic or anaprotic solvent, preferably it is a protic solvent such as an alcohole.g., methanol or ethanol, or an aqueous alcohol. The reaction may becarried out at temperatures greater than room temperature. Thetemperature is generally from about 20° C. to about 100° C., preferablyabout 40° C. to about 80° C. By “about” is meant that the temperaturerange is preferably within 10 degrees Celsius of the indicatedtemperature, more preferably within 5 degrees Celsius of the indicatedtemperature and, most preferably, within 2 degrees Celsius of theindicated temperature. Thus, for example, by “about 60° C.” is meant 60°C.±10° C., preferably 60° C.±5° C. and most preferably, 60° C.±2° C.

Suitable amines include alicyclic and cyclic secondary amines. Theseamines are either commercially available from Aldrich, Sigma, etc., orthey can be prepared by methods well known in the art. Exemplarysecondary amines include dimethylamine, diethylamine andbis(2-hydroxyethyl)amine. Exemplary cyclic secondary amines includeN-alkyl piperazine and pyrrolidine.

Compounds of formula II, where R^(3′) and R^(4′) combine to form aheteroaryl ring, may be prepared by reacting the parent3-pyrrolidinyl-2-indolinone (V) with a suitable aldehyde to yield anintermediate N-hydroxyalkyl derivative of V, and reacting theintermediate with phosphorus oxychloride and a suitable heteroaryl suchas pyridine.

The reaction may be carried out at temperatures less than roomtemperature. The temperature is generally from about −20° C. to about20° C., preferably about −10° C. to about 10° C. By “about” is meantthat the temperature range is preferably within 10 degrees Celsius ofthe indicated temperature, more preferably within 5 degrees Celsius ofthe indicated temperature and, most preferably, within 2 degrees Celsiusof the indicated temperature. Thus, for example, by “about 0° C.” ismeant 0° C.±10° C., preferably 0° C.±5° C. and most preferably, 0° C.±2°C.

Compounds of Formula III may be prepared from a compound of Formula V asshown below:

A compound of Formula III where R⁵′ is as defined in the Summary of theInvention can be readily prepared by acylating a compound of Formula Vwith a suitable agents e.g., carboxylic acid anhydrides such as aceticanhydride, succinic anhydride, carboxylic acid chlorides such as acetylchloride, butryl chloride, and the like or carboxylic acid activeesters. The reaction may be carried out in the presence of an organicbase, preferably a tertiary nitrogen base. Examples of tertiary nitrogenbases include, but are not limited to, trimethylamine, triethylamine,pyridine, and 1,8-diazabicyclo[5.4.1]undec-7-ene.

The solvent in which the reaction is carried out may be an aproticsolvent. A “protic solvent” is a solvent which has hydrogen atom(s)covalently bonded to oxygen or nitrogen atoms which renders the hydrogenatoms appreciably acidic and thus capable of being “shared” with asolute through hydrogen bonding. An “aprotic solvent” may be polar ornon-polar but, in either case, does not contain acidic hydrogens andtherefore is not capable of hydrogen bonding with solutes. Examples,without limitation, of non-polar aprotic solvents, are pentane, hexane,benzene, toluene, methylene chloride and carbon tetrachloride. Examplesof polar aprotic solvents are chloroform, tetrahydrofuran,dimethylsulfoxide, dimethylformamide and pyridine. In a presentlypreferred embodiment of this invention, the solvent is a polar aproticprotic solvent, preferably dimethylformamide, tetrahydrofuran orpyridine. The reaction is typically carried out at room temperature.

Compounds of Formula IV may be prepared from a compound of Formula V asshown below:

A compound of Formula IV where R²–R¹⁰ are as defined in the Summary ofthe Invention an R^(a) and R^(b) are not hydrogen can be prepared byreacting V with a phosphorylating agent such as phosphoryl halide suchas dimethyl chlorophosphate. The reaction is carried out in the presenceof a strong base such as sodium hydride and in an organic solvent suchas THF, DMF, and the like. The methyl groups can be removed undersuitable demethylation reaction conditions such as treatment withN,O-Bis(trimethylsilyl)acetamide in the presence oftrimetylsilylbromide. The reaction is carried out in a polar organicsolvent such as acetonitrile.

The preparation of compounds of Formula I–IV may further include thestep of removing a protecting group. “Protecting group” refers to agroup used to render a reactive moiety inert until removal of the group.Reactive moieties are well known to the skilled artisan; preferredreactive moieties include reactive nitrogen, oxygen, sulfur, carboxyland carbonyl groups. Exemplary nitrogen protecting groups include, butare not limited to, benzyl, benzyloxycarbonyl, tert-butoxycarbonyl,silyl groups (e.g., tert-butyldimethylsilyl),9-fluorenylmethoxycarbonyl, 9-phenyl-9-fluorenyl and arylsulfonyl groups(e.g., toluenesulfonyl). Exemplary oxygen protecting groups include, butare not limited to, allyloxycarbonyl, benzoyl, benzyl, tert-butyl, silylgroups (e.g., tert-butyldimethylsilyl), 2-ethoxyethyl, p-methoxybenzyl,methoxymethyl, pivaloyl, tetrahydropyran-2-yl and trityl. Exemplarycarboxyl protecting groups include, without limitation, methyl, allyl,benzyl, silyl groups (e.g., tert-butyldimethylsilyl) and p-nitrobenzyl.Exemplary carbonyl protecting groups include, but are not limited to,acetyl groups (e.g., O,O-acetals).

Protecting groups may be removed using methods known in the literature.For example, for the removal of nitrogen protecting groups see Greene etal. (1991) Protecting Groups in Organic Synthesis, 2^(nd) ed., JohnWiley & Sons, New York, pp. 309–405 and Kocienski (1994) ProtectingGroups, Thieme, New York, pp. 185–243. Methods for the removal ofparticular protecting groups are exemplified herein.

Utility

The PKs whose catalytic activity is modulated by the compounds of thisinvention include protein tyrosine kinases of which there are two types,receptor tyrosine kinases (RTKs) and cellular tyrosine kinases (CTKs),and serine-threonine kinases (STKs). RTK mediated signal transduction,is initiated by extracellular interaction with a specific growth factor(ligand), followed by receptor dimerization, transient stimulation ofthe intrinsic protein tyrosine kinase activity and phosphorylation.Binding sites are thereby created for intracellular signal transductionmolecules and lead to the formation of complexes with a spectrum ofcytoplasmic signaling molecules that facilitate the appropriate cellularresponse (e.g., cell division, metabolic effects on the extracellularmicroenvironment, etc.). See, Schlessinger and Ullrich, 1992, Neuron9:303–391.

It has been shown that tyrosine phosphorylation sites on growth factorreceptors function as high-affinity binding sites for SH2 (src homology)domains of signaling molecules. Fantl et al., 1992, Cell 69:413–423,Songyang et al., 1994, Mol. Cell. Biol. 14:2777–2785), Songyang et al.,1993, Cell 72:767–778, and Koch et al., 1991, Science 252:668–678.Several intracellular substrate proteins that associate with RTKs havebeen identified. They may be divided into two principal groups: (1)substrates that have a catalytic domain, and (2) substrates which lacksuch domain but which serve as adapters and associate with catalyticallyactive molecules. Songyang et al., 1993, Cell 72:767–778. Thespecificity of the interactions between receptors and SH2 domains oftheir substrates is determined by the amino acid residues immediatelysurrounding the phosphorylated tyrosine residue. Differences in thebinding affinities between SH2 domains and the amino acid sequencessurrounding the phosphotyrosine residues on particular receptors areconsistent with the observed differences in their substratephosphorylation profiles. Songyang et al., 1993, Cell 72:767–778. Theseobservations suggest that the function of each RTK is determined notonly by its pattern of expression and ligand availability but also bythe array of downstream signal transduction pathways that are activatedby a particular receptor. Thus, phosphorylation provides an importantregulatory step which determines the selectivity of signaling pathwaysrecruited by specific growth factor receptors, as well asdifferentiation factor receptors.

STKs, being primarily cytosolic, affect the internal biochemistry of thecell, often as a down-line response to a PTK event. STKs have beenimplicated in the signaling process which initiates DNA synthesis andsubsequent mitosis leading to cell proliferation.

Thus, PK signal transduction results in, among other responses, cellproliferation, differentiation, growth and metabolism. Abnormal cellproliferation may result in a wide array of disorders and diseases,including the development of neoplasia such as carcinoma, sarcoma,glioblastoma and hemangioma, disorders such as leukemia, psoriasis,arteriosclerosis, arthritis and diabetic retinopathy and other disordersrelated to uncontrolled angiogenesis and/or vasculogenesis.

In another aspect, the protein kinase, the catalytic activity of whichis modulated by contact with a compound of this invention, is a proteintyrosine kinase, more particularly, a receptor protein tyrosine kinase.Among the receptor protein tyrosine kinases whose catalytic activity canbe modulated with a compound of this invention, or salt thereof, are,without limitation, EGF, HER2, HER3, HER4, IR, IGF-1R, IRR, PDGFRα,PDGFRβ, CSFIR, C-Kit, C-fms, Flk-1R, Flk4, KDR/Flk-1, Flt-1, FGFR-1R,FGFR-2R, FGFR-3R and FGFR-4R.

The protein tyrosine kinase whose catalytic activity is modulated bycontact with a compound of this invention, or a salt thereof, can alsobe a non-receptor or cellular protein tyrosine kinase (CTK). Thus, thecatalytic activity of CTKs such as, without limitation, Src, Frk, Btk,Csk, Abl, ZAP70, Fes, Fps, Fak, Jak, Ack, Yes, Fyn, Lyn, Lck, Blk, Hck,Fgr and Yrk, may be modulated by contact with a compound or salt of thisinvention.

Still another group of PKs which may have their catalytic activitymodulated by contact with a compound of this invention are theserine-threonine protein kinases such as, without limitation, CDK2 andRaf.

In another aspect, this invention relates to a method for treating orpreventing a PK related disorder by administering a therapeuticallyeffective amount of a compound of this invention, or a salt thereof, toan organism.

It is also an aspect of this invention that a pharmaceutical compositioncontaining a compound of this invention, or a salt thereof, isadministered to an organism for the purpose of preventing or treating aPK related disorder.

This invention is therefore directed to compounds that modulate PKsignal transduction by affecting the enzymatic activity of RTKs, CTKsand/or STKs, thereby interfering with the signals transduced by suchproteins. More particularly, the present invention is directed tocompounds which modulate RTK, CTK and/or STK mediated signaltransduction pathways as a therapeutic approach to cure many kinds ofsolid tumors, including but not limited to carcinomas, sarcomasincluding Kaposi's sarcoma, erythroblastoma, glioblastoma, meningioma,astrocytoma, melanoma and myoblastoma. Treatment or prevention ofnon-solid tumor cancers such as leukemia are also contemplated by thisinvention. Indications may include, but are not limited to braincancers, bladder cancers, ovarian cancers, gastric cancers, pancreaticcancers, colon cancers, blood cancers, lung cancers and bone cancers.

Further examples, without limitation, of the types of disorders relatedto inappropriate PK activity that the compounds described herein may beuseful in preventing, treating and studying, are cell proliferativedisorders, fibrotic disorders, metabolic disorders and infectiousdiseases.

Cell proliferative disorders, which may be prevented, treated or furtherstudied by the present invention include cancer, blood vesselproliferative disorders and mesangial cell proliferative disorders.

Blood vessel proliferative disorders refer to disorders related toabnormal vasculogenesis (blood vessel formation) and angiogenesis(spreading of blood vessels). While vasculogenesis and angiogenesis playimportant roles in a variety of normal physiological processes such asembryonic development, corpus luteum formation, wound healing and organregeneration, they also play a pivotal role in cancer development wherethey result in the formation of new capillaries needed to keep a tumoralive. Other examples of blood vessel proliferation disorders includearthritis, where new capillary blood vessels invade the joint anddestroy cartilage, and ocular diseases, like diabetic retinopathy, wherenew capillaries in the retina invade the vitreous, bleed and causeblindness.

Two structurally related RTKs have been identified to bind VEGF withhigh affinity: the fms-like tyrosine 1 (fit-I) receptor (Shibuya et al.,1990, Oncogene, 5:519–524; De Vries et al., 1992, Science, 255:989–991)and the KDR/FLK-1 receptor, also known as VEGF-R2. Vascular endothelialgrowth factor (VEGF) has been reported to be an endothelial cellspecific mitogen with in vitro endothelial cell growth promotingactivity. Ferrara & Henzel, 1989, Biochem. Biophys. Res. Comm.,161:851–858; Vaisman et al., 1990, J. Biol. Chem., 265:19461–19566.Information set forth in U.S. application Ser. Nos. 08/193,829,08/038,596 and 07/975,750, strongly suggest that VEGF is not onlyresponsible for endothelial cell proliferation, but also is the primeregulator of normal and pathological angiogenesis. See generally,Klagsburn & Soker, 1993, Current Biology, 3(10)699–702; Houck, et al.,1992, J. Biol. Chem., 267:26031–26037.

Normal vasculogenesis and angiogenesis play important roles in a varietyof physiological processes such as embryonic development, wound healing,organ regeneration and female reproductive processes such as follicledevelopment in the corpus luteum during ovulation and placental growthafter pregnancy. Folkman & Shing, 1992, J. Biological Chem.,267(16):10931–34. Uncontrolled vasculogenesis and/or angiogenesis hasbeen associated with diseases such as diabetes as well as with malignantsolid tumors that rely on vascularization for growth. Klagsburn & Soker,1993, Current Biology, 3(10):699–702; Folkham, 1991, J. Natl. CancerInst., 82:4–6; Weidner, et al., 1991, New Engl. J. Med., 324:1–5.

As presently understood, the role of VEGF in endothelial cellproliferation and migration during angiogenesis and vasculogenesisindicates an important role for the KDR/FLK-1 receptor in theseprocesses. Diseases such as diabetes mellitus (Folkman, 198, in XIthCongress of Thrombosis and Haemostasis (Verstraeta, et al., eds.), pp.583–596, Leuven University Press, Leuven) and arthritis, as well asmalignant tumor growth may result from uncontrolled angiogenesis. Seee.g., Folkman, 1971, N. Engl. J. Med., 285:1182–1186. The receptors towhich VEGF specifically binds are an important and powerful therapeutictarget for the regulation and modulation of vasculogenesis and/orangiogenesis and a variety of severe diseases which involve abnormalcellular growth caused by such processes. Plowman, et al., 1994, DN&P,7(6):334–339. More particularly, the KDR/FLK-1 receptor's highlyspecific role in neovascularization make it a choice target fortherapeutic approaches to the treatment of cancer and other diseaseswhich involve the uncontrolled formation of blood vessels.

Thus, one aspect of the present invention relates to compounds capableof regulating and/or modulating tyrosine kinase signal transductionincluding KDR/FLK-1 receptor signal transduction in order to inhibit orpromote angiogenesis and/or vasculogenesis, that is, compounds thatinhibit, prevent, or interfere with the signal transduced by KDR/FLK-1when activated by ligands such as VEGF. Although it is believed that thecompounds of the present invention act on a receptor or other componentalong the tyrosine kinase signal transduction pathway, they may also actdirectly on the tumor cells that result from uncontrolled angiogenesis.

Although the nomenclature of the human and murine counterparts of thegeneric “flk-I” receptor differ, they are, in many respects,interchangeable. The murine receptor, Flk-1, and its human counterpart,KDR, share a sequence homology of 93.4% within the intracellular domain.Likewise, murine FLK-I binds human VEGF with the same affinity as mouseVEGF, and accordingly, is activated by the ligand derived from eitherspecies. Millauer et al., 1993, Cell, 72:835–846; Quinn et al., 1993,Proc. Natl. Acad. Sci. USA, 90:7533–7537. FLK-1 also associates with andsubsequently tyrosine phosphorylates human RTK substrates (e.g., PLC-γor p85) when co-expressed in 293 cells (human embryonal kidneyfibroblasts).

Models which rely upon the FLK-1 receptor therefore are directlyapplicable to understanding the KDR receptor. For example, use of themurine FLK-1 receptor in methods which identify compounds that regulatethe murine signal transduction pathway are directly applicable to theidentification of compounds which may be used to regulate the humansignal transduction pathway, that is, which regulate activity related tothe KDR receptor. Thus, chemical compounds identified as inhibitors ofKDR/FLK-1 in vitro, can be confirmed in suitable in vivo models. Both invivo mouse and rat animal models have been demonstrated to be ofexcellent value for the examination of the clinical potential of agentsacting on the KDR/FLK-1 induced signal transduction pathway.

Thus, in one aspect, this invention is directed to compounds thatregulate, modulate and/or inhibit vasculogenesis and/or angiogenesis byaffecting the enzymatic activity of the KDR/FLK-1 receptor andinterfering with the signal transduced by KDR/FLK-1. In another aspect,the present invention is directed to compounds which regulate, modulateand/or inhibit the KDR/FLK-1 mediated signal transduction pathway as atherapeutic approach to the treatment of many kinds of solid tumorsincluding, but not limited to, glioblastoma, melanoma and Kaposi'ssarcoma, and ovarian, lung, mammary, prostate, pancreatic, colon andepidermoid carcinoma. In addition, data suggest the administration ofcompounds which inhibit the KDR/Flk-1 mediated signal transductionpathway may also be used in the treatment of hemangioma, restenois anddiabetic retinopathy.

A further aspect of this invention relates to the inhibition ofvasculogenesis and angiogenesis by other receptor-mediated pathways,including the pathway comprising the flt-1 receptor.

Receptor tyrosine kinase mediated signal transduction is initiated byextracellular interaction with a specific growth factor (ligand),followed by receptor dimerization, transient stimulation of theintrinsic protein tyrosine kinase activity and autophosphorylation.Binding sites are thereby created for intracellular signal transductionmolecules which leads to the formation of complexes with a spectrum ofcytoplasmic signaling molecules that facilitate the appropriate cellularresponse, e.g., cell division and metabolic effects to the extracellularmicroenvironment. See, Schlessinger and Ullrich, 1992, Neuron, 9:1–20.

The close homology of the intracellular regions of KDR/FLK-1 with thatof the PDGF-β receptor (50.3% homology) and/or the related flt-1receptor indicates the induction of overlapping signal transductionpathways. For example, for the PDGF-β receptor, members of the srcfamily (Twamley et al., 1993, Proc. Natl. Acad. Sci. USA, 90:7696–7700),phosphatidylinositol-3′-kinase (Hu et al., 1992, Mol. Cell. Biol.,12:981–990), phospholipase cγ (Kashishian & Cooper, 1993, Mol. Cell.Biol., 4:49–51), ras-GTPase-activating protein, (Kashishian et al.,1992, EMBO J., 11:1373–1382), PTP-ID/syp (Kazlauskas et al., 1993, Proc.Natl. Acad. Sci. USA, 10 90:6939–6943), Grb2 (Arvidsson et al., 1994,Mol. Cell. Biol., 14:6715–6726), and the adapter molecules Shc and Nck(Nishimura et al., 1993, Mol. Cell. Biol., 13:6889–6896), have beenshown to bind to regions involving different autophosphorylation sites.See generally, Claesson-Welsh, 1994; Prog. Growth Factor Res., 5:37–54.Thus, it is likely that signal transduction pathways activated byKDR/FLK-1 include the ras pathway (Rozakis et al., 1992, Nature,360:689–692), the PI-3′-kinase, the src-mediated and the plcγ-mediatedpathways. Each of these pathways may play a critical role in theangiogenic and/or vasculogenic effect of KDR/FLK-1 in endothelial cells.Consequently, a still further aspect of this invention relates to theuse of the organic compounds described herein to modulate angiogenesisand vasculogenesis as such processes are controlled by these pathways.

Conversely, disorders related to the shrinkage, contraction or closingof blood vessels, such as restenosis, are also implicated and may betreated or prevented by the methods of this invention.

Fibrotic disorders refer to the abnormal formation of extracellularmatrices. Examples of fibrotic disorders include hepatic cirrhosis andmesangial cell proliferative disorders. Hepatic cirrhosis ischaracterized by the increase in extracellular matrix constituentsresulting in the formation of a hepatic scar. An increased extracellularmatrix resulting in a hepatic scar can also be caused by a viralinfection such as hepatitis. Lipocytes appear to play a major role inhepatic cirrhosis. Other fibrotic disorders implicated includeatherosclerosis.

Mesangial cell proliferative disorders refer to disorders brought aboutby abnormal proliferation of mesangial cells. Mesangial proliferativedisorders include various human renal diseases such asglomerulonephritis, diabetic nephropathy and malignant nephrosclerosisas well as such disorders as thrombotic microangiopathy syndromes,transplant rejection, and glomerulopathies. The RTK PDGFR has beenimplicated in the maintenance of mesangial cell proliferation. Floege etal., 1993, Kidney International 43:47S–54S.

Many cancers are cell proliferative disorders and, as noted previously,PKs have been associated with cell proliferative disorders. Thus, it isnot surprising that PKs such as, for example, members of the RTK familyhave been associated with the development of cancer. Some of thesereceptors, like EGFR (Tuzi et al., 1991, Br. J. Cancer 63:227–233, Torpet al., 1992, APMIS 100:713–719) HER2/neu (Slamon et al., 1989, Science244:707–712) and PDGF-R (Kumabe et al., 1992, Oncogene, 7:627–633) areover-expressed in many tumors and/or persistently activated by autocrineloops. In fact, in the most common and severe cancers these receptorover-expressions (Akbasak and Suner-Akbasak et al., 1992, J. Neurol.Sci., 111:119–133, Dickson et al., 1992, Cancer Treatment Res.61:249–273, Korc et al., 1992, J. Clin. Invest. 90:1352–1360) andautocrine loops (Lee and Donoghue, 1992, J. Cell. Biol., 118:1057–1070,Korc et al., supra, Akbasak and Suner-Akbasak et al., supra) have beendemonstrated. For example, EGFR has been associated with squamous cellcarcinoma, astrocytoma, glioblastoma, head and neck cancer, lung cancerand bladder cancer. HER2 has been associated with breast, ovarian,gastric, lung, pancreatic and bladder cancer. PDGFR has been associatedwith glioblastoma and melanoma as well as lung, ovarian and prostatecancer. The RTK c-met has also been associated with malignant tumorformation. For example, c-met has been associated with, among othercancers, colorectal, thyroid, pancreatic, gastric and hepatocellularcarcinomas and lymphomas. Additionally c-met has been linked toleukemia. Over-expression of the c-met gene has also been detected inpatients with Hodgkins disease and Burkitts disease.

IGF-IR, in addition to being implicated in nutritional support and intype-II diabetes, has also been associated with several types ofcancers. For example, IGF-I has been implicated as an autocrine growthstimulator for several tumor types, e.g. human breast cancer carcinomacells (Arteaga et al., 1989, J. Clin. Invest. 84:1418–1423) and smalllung tumor cells (Macauley et al., 1990, Cancer Res., 50:2511–2517). Inaddition, IGF-I, while integrally involved in the normal growth anddifferentiation of the nervous system, also appears to be an autocrinestimulator of human gliomas. Sandberg-Nordqvist et al., 1993, CancerRes. 53:2475–2478. The importance of IGF-IR and its ligands in cellproliferation is further supported by the fact that many cell types inculture (fibroblasts, epithelial cells, smooth muscle cells,T-lymphocytes, myeloid cells, chondrocytes and osteoblasts (the stemcells of the bone marrow)) are stimulated to grow by IGF-I. Goldring andGoldring, 1991, Eukaryotic Gene Expression, 1:301–326. In a series ofrecent publications, Baserga suggests that IGF-IR plays a central rolein the mechanism of transformation and, as such, could be a preferredtarget for therapeutic interventions for a broad spectrum of humanmalignancies. Baserga, 1995, Cancer Res., 55:249–252, Baserga, 1994,Cell 79:927–930, Coppola et al., 1994, Mol. Cell. Biol., 14:4588–4595.

STKs have been implicated in many types of cancer including, notably,breast cancer. (Cance, et al., Int. J. Cancer, 54:571–77 (1993)).

The association between abnormal PK activity and disease is notrestricted to cancer. For example, RTKs have been associated withdiseases such as psoriasis, diabetes mellitus, endometriosis,angiogenesis, atheromatous plaque development, Alzheimer's disease, vonHippel-Lindau disease, epidermal hyperproliferation, neurodegenerativediseases, age-related macular degeneration and hemangiomas. For example,EGFR has been indicated in corneal and dermal wound healing. Defects inInsulin-R and IGF-1R are indicated in type-II diabetes mellitus. A morecomplete correlation between specific RTKs and their therapeuticindications is set forth in Plowman et al., 1994, DN&P 7:334–339.

As noted previously, CTKs including, but not limited to, src, abl, fps,yes, fyn, lyn, lck, blk, hck, fgr and yrk (reviewed by Bolen et al.,1992, FASEB J., 6:3403–3409), are also involved in the proliferative andmetabolic signal transduction pathway and thus could be expected, andhave been shown, to be involved in many PTK-mediated disorders to whichthe present invention is directed. For example, mutated src (v-src) hasbeen shown to be an oncoprotein (pp60^(v-src)) in chicken. Moreover, itscellular homolog, the proto-oncogene pp60^(c-src) transmits oncogenicsignals of many receptors. Over-expression of EGFR or HER2/neu in tumorsleads to the constitutive activation of pp60^(c-src), which ischaracteristic of malignant cells but absent in normal cells. On theother hand, mice deficient in the expression of c-src exhibit anosteopetrotic phenotype, indicating a key participation of c-src inosteoclast function and a possible involvement in related disorders.

Similarly, Zap70 has been implicated in T-cell signaling which mayrelate to autoimmune disorders.

STKs have been associated with inflammation, autoimmune disease,immunoresponses, and hyperproliferation disorders such as restenosis,fibrosis, psoriasis, osteoarthritis and rheumatoid arthritis.

PKs have also been implicated in embryo implantation. Thus, thecompounds of this invention may provide an effective method ofpreventing such embryo implantation and thereby be useful as birthcontrol agents.

In yet another aspect, the compounds of the instant invention can alsobe used as anti-infective agents. For example, indolinone compounds areknown to exhibit antibacterial and antifungal activities. See, e.g.,Singh and Jha (1989) “Indolinone derivatives as potential antimicrobialagents,” Zentralbl. Mikrobiol. 144(2):105–109. In addition, indolinonecompounds have been reported to exhibit significant antiviral activity.See, e.g., Maass et al. (1993) “Viral resistance to thethiazolo-iso-indolinones, a new class of nonnucleoside inhibitors ofhuman immunodeficiency virus type 1 reverse transcriptase,” Antimicrob.Agents Chemother. 37(12):2612–2617.

Finally, both RTKs and CTKs are currently suspected as being involved inhyperimmune disorders.

A method for identifying a chemical compound that modulates thecatalytic activity of one or more of the above discussed protein kinasesis another aspect of this invention. The method involves contactingcells expressing the desired protein kinase with a compound of thisinvention (or its salt) and monitoring the cells for any effect that thecompound has on them. The effect may be any observable, either to thenaked eye or through the use of instrumentation, change or absence ofchange in a cell phenotype. The change or absence of change in the cellphenotype monitored may be, for example, without limitation, a change orabsence of change in the catalytic activity of the protein kinase in thecells or a change or absence of change in the interaction of the proteinkinase with a natural binding partner.

Pharmaceutical Compositions and Administration

A compound of the present invention or a physiologically acceptable saltthereof, can be administered as such to a human patient or can beadministered in pharmaceutical compositions in which the foregoingmaterials are mixed with suitable carriers or excipient(s). Techniquesfor formulation and administration of drugs may be found in “Remington'sPharmacological Sciences,” Mack Publishing Co., Easton, Pa., latestedition.

Routes of Administration

As used herein, “administer” or “administration” refers to the deliveryof a compound or salt of the present invention or of a pharmaceuticalcomposition containing a compound or salt of this invention to anorganism for the purpose of prevention or treatment of a PK-relateddisorder.

Suitable routes of administration may include, without limitation, oral,rectal, transmucosal or intestinal administration or intramuscular,subcutaneous, intramedullary, intrathecal, direct intraventricular,intravenous, intravitreal, intraperitoneal, intranasal, or intraocularinjections. The preferred routes of administration are oral andparenteral.

Alternatively, one may administer the compound in a local rather thansystemic manner, for example, via injection of the compound directlyinto a solid tumor, often in a depot or sustained release formulation.

Furthermore, one may administer the drug in a targeted drug deliverysystem, for example, in a liposome coated with tumor-specific antibody.The liposomes will be targeted to and taken up selectively by the tumor.

Composition/Formulation

Pharmaceutical compositions of the present invention may be manufacturedby processes well known in the art, e.g., by means of conventionalmixing, dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping, lyophilizing processes or spray drying.

Pharmaceutical compositions for use in accordance with the presentinvention may be formulated in conventional manner using one or morephysiologically acceptable carriers comprising excipients andauxiliaries which facilitate processing of the active compounds intopreparations which can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

For injection, the compounds of the invention may be formulated inaqueous solutions, preferably in physiologically compatible buffers suchbuffers with or without a low concentration of surfactant or cosolvent,or physiological saline buffer. For transmucosal administration,penetrants appropriate to the barrier to be permeated are used in theformulation. Such penetrants are generally known in the art.

For oral administration, the compounds can be formulated by combiningthe active compounds with pharmaceutically acceptable carriers wellknown in the art. Such carriers enable the compounds of the invention tobe formulated as tablets, pills, lozenges; dragees, capsules, liquids,gels, syrups, slurries, suspensions and the like, for oral ingestion bya patient. Pharmaceutical preparations for oral use can be made using asolid excipient, optionally grinding the resulting mixture, andprocessing the mixture of granules, after adding other suitableauxiliaries if desired, to obtain tablets or dragee cores. Usefulexcipients are, in particular, fillers such as sugars, includinglactose, sucrose, mannitol, or sorbitol, cellulose preparations such as,for example, maize starch, wheat starch, rice starch and potato starchand other materials such as gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinyl-pyrrolidone (PVP). If desired, disintegrating agents may beadded, such as cross-linked polyvinyl pyrrolidone, agar, or alginicacid. A salt such as sodium alginate may also be used.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical compositions which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with a fillersuch as lactose, a binder such as starch, and/or a lubricant such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, liquid polyethyleneglycols, cremophor, capmul, medium or long chain mono- di- ortriglycerides. Stabilizers may be added in these formulations, also.

For administration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of an aerosolspray using a pressurized pack or a nebulizer and a suitable propellant,e.g., without limitation, dichlorodifluoromethane,trichlorofluoromethane, dichlorotetra-fluoroethare or carbon dioxide. Inthe case of a pressurized aerosol, the dosage unit may be controlled byproviding a valve to deliver a metered amount. Capsules and cartridgesof, for example, gelatin for use in an inhaler or insufflator may beformulated containing a powder mix of the compound and a suitable powderbase such as lactose or starch.

The compounds may also be formulated for parenteral administration,e.g., by bolus injection or continuous infusion. Formulations forinjection may be presented in unit dosage form, e.g., in ampoules or inmulti-dose containers, with an added preservative. The compositions maytake such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulating materials such assuspending, stabilizing and/or dispersing agents.

Pharmaceutical compositions for parenteral administration includeaqueous solutions of a water soluble form, such as, without limitation,a salt, of the active compound. Additionally, suspensions of the activecompounds may be prepared in a lipophilic vehicle. Suitable lipophilicvehicles include fatty oils such as sesame oil, synthetic fatty acidesters such as ethyl oleate and triglycerides, or materials such asliposomes. Aqueous injection suspensions may contain substances whichincrease the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Optionally, the suspension may alsocontain suitable stabilizers and/or agents that increase the solubilityof the compounds to allow for the preparation of highly concentratedsolutions.

Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile, pyrogen-free waterwith or without additional surfactants or cosolvents such as polysorbate80, Cremophor, cyclodextrin sulfobutyl ether, propylene glycol, orpolyethylene glycol such as PEG-300 or PEG-400, before use.

The compounds may also be formulated in rectal compositions such assuppositories or retention enemas, using, e.g., conventional suppositorybases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds mayalso be formulated as depot preparations. Such long acting formulationsmay be administered by implantation (for example, subcutaneously orintramuscularly) or by intramuscular injection. A compound of thisinvention may be formulated for this route of administration withsuitable polymeric or hydrophobic materials (for instance, in anemulsion with a pharmacologically acceptable oil), with ion exchangeresins, or as a sparingly soluble derivative such as, withoutlimitation, a sparingly soluble salt.

A non-limiting example of a pharmaceutical carrier for the hydrophobiccompounds of the invention is a cosolvent system comprising benzylalcohol, a nonpolar surfactant, a water-miscible organic polymer and anaqueous phase such as the VPD co-solvent system. VPD is a solution of 3%w/v benzyl alcohol, 8% w/v of the nonpolar surfactant Polysorbate 80™,and 65% w/v polyethylene glycol 300, made up to volume in absoluteethanol. The VPD co-solvent system (VPD:D5W) consists of VPD diluted 1:1with a 5% dextrose in water solution. This co-solvent system dissolveshydrophobic compounds well, and itself produces low toxicity uponsystemic administration. Naturally, the proportions of such a co-solventsystem may be varied considerably without destroying its solubility andtoxicity characteristics. Furthermore, the identity of the co-solventcomponents may be varied: for example, other low-toxicity nonpolarsurfactants may be used instead of Polysorbate 80™, the fraction size ofpolyethylene glycol may be varied, other biocompatible polymers mayreplace polyethylene glycol, e.g., polyvinyl pyrrolidone, and othersugars or polysaccharides may substitute for dextrose.

Alternatively, other delivery systems for hydrophobic pharmaceuticalcompounds may be employed. Liposomes and emulsions are well knownexamples of delivery vehicles or carriers for hydrophobic drugs. Inaddition, certain organic solvents such as dimethylsulfoxide also may beemployed, although often at the cost of greater toxicity.

Additionally, the compounds may be delivered using a sustained-releasesystem, such as semipermeable matrices of solid hydrophobic polymerscontaining the therapeutic agent. Various sustained-release materialshave been established and are well known by those skilled in the art.Sustained-release capsules may, depending on their chemical nature,release the compounds for a few weeks up to over 100 days. Depending onthe chemical nature and the biological stability of the therapeuticreagent, additional strategies for protein stabilization may beemployed.

The pharmaceutical compositions herein also may comprise suitable solidor gel phase carriers or excipients. Examples of such carriers orexcipients include, but are not limited to, calcium carbonate, calciumphosphate, various sugars, starches, cellulose derivatives, gelatin, andpolymers such as polyethylene glycols.

Many of the PK modulating compounds of the invention may be provided asphysiologically acceptable salts wherein the claimed compound may formthe negatively or the positively charged species. Examples of salts inwhich the compound forms the positively charged moiety include, withoutlimitation, quaternary ammonium (defined elsewhere herein), salts suchas the hydrochloride, sulfate, citrate, mesylate, lactate, tartrate,maleate, succinate wherein the nitrogen atom of the quaternary ammoniumgroup is a nitrogen of the selected compound of this invention which hasreacted with the appropriate acid. Salts in which a compound of thisinvention forms the negatively charged species include, withoutlimitation, the sodium, potassium, calcium and magnesium salts formed bythe reaction of a carboxylic acid group in the compound with anappropriate base (e.g. sodium hydroxide (NaOH), potassium hydroxide(KOH), Calcium hydroxide (Ca(OH)₂), etc.).

Dosage

Pharmaceutical compositions suitable for use in the present inventioninclude compositions wherein the active ingredients are contained in anamount sufficient to achieve the intended purpose, i.e., the modulationof PK activity or the treatment or prevention of a PK-related disorder.

More specifically, a therapeutically effective amount means an amount ofcompound effective to prevent, alleviate or ameliorate symptoms ofdisease or prolong the survival of the subject being treated.Therapeutically effective amounts of compounds of Formula I–IV may rangefrom approximately 10 mg/m² to 400 mg/m², preferably 50 mg/m² to 300mg/m², more preferably 100 mg/m² to 220 mg/m², even more preferably 195mg/m². For any compound used in the methods of the invention, thetherapeutically effective amount or dose can be estimated initially fromcell culture assays. Then, the dosage can be formulated for use inanimal models so as to achieve a circulating concentration range thatincludes the IC₅₀ as determined in cell culture (i.e., the concentrationof the test compound which achieves a half-maximal inhibition of the PKactivity). Such information can then be used to more accuratelydetermine useful doses in humans.

Toxicity and therapeutic efficacy of the compounds described herein canbe determined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., by determining the IC₅₀ and the LD₅₀ (bothof which are discussed elsewhere herein) for a subject compound. Thedata obtained from these cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage mayvary depending upon the dosage form employed and the route ofadministration utilized. The exact formulation, route of administrationand dosage can be chosen by the individual physician in view of thepatient's condition. (See e.g., Fingl, et al., 1975, in “ThePharmacological Basis of Therapeutics”, Ch. 1 p. 1).

Dosage amount and interval may be adjusted individually to provideplasma levels of the active species which are sufficient to maintain thekinase modulating effects. These plasma levels are referred to asminimal effective concentrations (MECs). The MEC will vary for eachcompound but can be estimated from in vitro data, e.g., theconcentration necessary to achieve 50–90% inhibition of a kinase may beascertained using the assays described herein. Preferably, the Dosagesnecessary to achieve the MEC will depend on individual characteristicsand route of administration. HPLC assays or bioassays can be used todetermine plasma concentrations.

Dosage intervals can also be determined using MEC value. Compoundsshould be administered using a regimen that maintains plasma levelsabove the MEC for 10–90% of the time, preferably between 30–90% and mostpreferably between 50–90%. In cases of local administration or selectiveuptake, the effective local concentration of the drug may not be relatedto plasma concentration and other procedures known in the art may beemployed to determine the correct dosage amount and interval.

The amount of a composition administered will, of course, be dependenton the subject being treated, the severity of the affliction, the mannerof administration, the judgment of the prescribing physician, etc.

Packaging

The compositions may, if desired, be presented in a pack or dispenserdevice, such as an FDA approved kit, which may contain one or more unitdosage forms containing the active ingredient. The pack may for examplecomprise metal or plastic foil, such as a blister pack. The pack ordispenser device may be accompanied by instructions for administration.The pack or dispenser may also be accompanied by a notice associatedwith the container in a form prescribed by a governmental agencyregulating the manufacture, use or sale of pharmaceuticals, which noticeis reflective of approval by the agency of the form of the compositionsor of human or veterinary administration. Such notice, for example, maybe of the labeling approved by the U.S. Food and Drug Administration forprescription drugs or of an approved product insert. Compositionscomprising a compound of the invention formulated in a compatiblepharmaceutical carrier may also be prepared, placed in an appropriatecontainer, and labeled for treatment of an indicated condition. Suitableconditions indicated on the label may include treatment of a tumor,inhibition of angiogenesis, treatment of fibrosis, diabetes, and thelike.

It is also an aspect of this invention that a compound described herein,or its salt, might be combined with other chemotherapeutic agents forthe treatment of the diseases and disorders discussed above. Forinstance, a compound or salt of this invention might be combined withalkylating agents such as fluorouracil (5-FU) alone or in furthercombination with leukovorin; or other alkylating agents such as, withoutlimitation, other pyrimidine analogs such as UFT, capecitabine,gemcitabine and cytarabine, the alkyl sulfonates, e.g., busulfan (usedin the treatment of chronic granulocytic leukemia), improsulfan andpiposulfan; aziridines, e.g., benzodepa, carboquone, meturedepa anduredepa; ethyleneimines and methylmelamines, e.g., altretamine,triethylenemelamine, triethylenephosphoramide,triethylenethiophosphoramide and trimethylolmelamine; and the nitrogenmustards, e.g., chlorambucil (used in the treatment of chroniclymphocytic leukemia, primary macroglobulinemia and non-Hodgkin'slymphoma), cyclophosphamide (used in the treatment of Hodgkin's disease,multiple myeloma, neuroblastoma, breast cancer, ovarian cancer, lungcancer, Wilm's tumor and rhabdomyosarcoma), estramustine, ifosfamide,novembrichin, prednimustine and uracil mustard (used in the treatment ofprimary thrombocytosis, non-Hodgkin's lymphoma, Hodgkin's disease andovarian cancer); and triazines, e.g., dacarbazine (used in the treatmentof soft tissue sarcoma).

Likewise a compound or salt of this invention might be expected to havea beneficial effect in combination with other antimetabolitechemotherapeutic agents such as, without limitation, folic acid analogs,e.g. methotrexate (used in the treatment of acute lymphocytic leukemia,choriocarcinoma, mycosis fungiodes breast cancer, head and neck cancerand osteogenic sarcoma) and pteropterin; and the purine analogs such asmercaptopurine and thioguanine which find use in the treatment of acutegranulocytic, acute lymphocytic and chronic granulocytic leukemias.

A compound or salt of this invention might also be expected to proveefficacious in combination with natural product based chemotherapeuticagents such as, without limitation, the vinca alkaloids, e.g.,vinblastin (used in the treatment of breast and testicular cancer),vincristine and vindesine; the epipodophylotoxins, e.g., etoposide andteniposide, both of which are useful in the treatment of testicularcancer and Kaposi's sarcoma; the antibiotic chemotherapeutic agents,e.g., daunorubicin, doxorubicin, epirubicin, mitomycin (used to treatstomach, cervix, colon, breast, bladder and pancreatic cancer),dactinomycin, temozolomide, plicamycin, bleomycin (used in the treatmentof skin, esophagus and genitourinary tract cancer); and the enzymaticchemotherapeutic agents such as L-asparaginase.

In addition to the above, a compound or salt of this invention might beexpected to have a beneficial effect used in combination with theplatinum coordination complexes (cisplatin, etc.); substituted ureassuch as hydroxyurea; methylhydrazine derivatives, e.g., procarbazine;adrenocortical suppressants, e.g., mitotane, aminoglutethimide; andhormone and hormone antagonists such as the adrenocorticosteriods (e.g.,prednisone), progestins (e.g., hydroxyprogesterone caproate); estrogens(e g., diethylstilbesterol); antiestrogens such as tamoxifen; androgens,e.g., testosterone propionate; and aromatase inhibitors (such asanastrozole).

Finally, the combination of a compound of this invention might beexpected to be particularly effective in combination with CAMPTOSAR™,GLEEVEC™, HERCEPTIN™, ENDOSTATIN™, Cox-2 inhibitors, MITOXANTRONE™ orPACLITAXEL™ for the treatment of solid tumor cancers or leukemias suchas, without limitation, acute myelogenous (non-lymphocytic) leukemia

EXAMPLES

The following preparations and examples are given to enable thoseskilled in the art to more clearly understand and to practice thepresent invention. They should not be considered as limiting the scopeof the invention, but merely as being illustrative and representativethereof.

In general HPLC data was obtained with a Zorbax SB C18 column (4.6 mmID×7.5 cm), a Perkin Elmer series 200 pump programmed to run from 10%acetonitrile/water 0.1% TFA (solvent A) to 90% acetonitrile/water(solvent B) with a flow rate of 1.5 mL/min. After 0.1 min on solvent A,a 5 min linear program to solvent B was run, followed by 3 min onsolvent B, before recycling to solvent A (2 min). Detection was with aPerkin Elmer diode array detector recording at 215 and 280 nM). NMRspectra were recorded on a Bruker instrument at 300 MHz.

Synthetic Examples

A. Oxymethyl Substituted Indolinones (I)

Example 1 Synthesis of(3Z)-3-[(3,5-dimethyl-1H-pyrrol-2-yl)-methylidene]-1-(hydroxymethyl)-1,3-dihydro-2H-indol-2-one(1)

Aqueous formaldehyde (15.0 g of 38% solution, 190 mmol) was added to astirred solution of3-(3,5-dimethyl-1H-pyrrol-2-ylmethylidene)-1,3-dihydro-indol-2-one (23.8g, 100 mmol) and triethylamine (15.0 g, 150 mmol) in dimethylformamide(200 mL). After 1 h, the solution was diluted with water and theprecipitate was filtered off, washed with water, and dried to give 26.4g of the title compound, mp 196–200° C. HPLC Rt 5.71 min. ¹H NMR (CDCl₃)δ 2.34 (s, 6H), 3.14 (t, 1H), 5.44 (d, 2H), 5.98 (d, 1H), 7.08 (m, 2H),7.18 (m, 1H), 7.36 (s, 1H), 7.48 (dd, 1H) and 13.0 (br s, 1H). Anal.Calcd for C₁₆H₁₆N₂O₂: C, 71.62; H, 6.01; N, 10.44. Found: C, 71.33; H,6.09; N, 10.43. A sample was recrystallized from ethyl acetate; mp200–202° C.

Example 2 Synthesis of {3(Z)-3-[(3,5-dimethyl-1Hpyrrol-2-yl)-methylidene]-2-oxo-2,3-dihydro-1H-indol-1-yl}methyl acetate(2)

Acetic anhydride (5.0 mL, 50 mmol) was added to a stirred solution of(3Z)-3-[(3,5-dimethyl-1H-pyrrol-2-yl)-methylidene]-1-(hydroxymethyl)-1,3-dihydro-2H-indol-2-one(1) (2.68 g, 10 mmol) in pyridine (30 mL). HPLC showed that acetylationwas complete in 1 h, at which time the solution was diluted with water(80 mL). The precipitate was filtered off, washed with water, and driedto give 2.94 g of the title compound, mp 145–152° C. The product wasrecrystallized from methanol to give 2.75 g, mp 150–152° C. HPLC Rt 6.58min. ¹H NMR (CDCl₃) δ 2.09 (s, 3H), 2.33 (s, 3H), 2.38 (s, 3H), 5.94 (s,2H), 5.99 (d, 1H), 7.10 (m, 2H), 7.20 (m, 1H), 7.39 (s, 1H), 7.50 (dd,1H) and 13.0 (br s, 1H). Anal. Calcd for C₁₈H₁₈N₂O₃: C, 69.66; H, 5.85;N, 9.03. Found: C, 69.57; H, 5.85; N, 9.06.

Example 3 Synthesis of4-({(3Z)-3-[(3,5-dimethyl-1H-pyrrol-2-yl)-methylidene]-2-oxo-2,3-dihydro-1H-indol-1-yl}methoxy)-4-oxobutanoicacid (3)

Succinic anhydride (5.0 g, 50 mmol) was added to a stirred solution of(3Z)-3-[(3,5-dimethyl-1H-pyrrol-2-yl)-methylidene]-1-(hydroxymethyl)-1,3-dihydro-2H-indol-2-one(1) (2.68 g, 10 mmol) in pyridine (30 mL). HPLC showed that the reactionwas 90% complete after 18 h. At this time, the solution was cooled to−10° C., the precipitate was filtered off, washed with aqueous pyridineand then with water, and was dried to give 3.57 g of the pyridinium saltof the title compound, mp 200–202° C. HPLC Rt 5.85 min. ¹H NMR (CDCl₃) δ2.35 (s, 3H), 2.40 (s, 3H), 2.70 (s, 4H), 6.01 (s, 3H), 7.10 (m, 2H),7.18 (m, 1H), 7.43 (s, 1H), 7.50(m, 3H), 7.93, (m, 1H), 8.68 (m, 1 H).HPLC Rt 5.85 min. ¹H NMR [(CD₃)₂SO] δ 2.33 (s, 3H), 2.36 (s, 3H), 2.50(m, 4H), 5.91 (s, 2H), 6.07 (d, 1H), 7.15 (m, 3H), 7.40 (m, 2H), 7.67(s, 1H), 7.80 (m, 2H), 8.58, (dd, 1H), 12.2 (s, 1 H), and 13.1 (s, 1H).Anal. Calcd for C₂₀H₂₀N₂O₅.C₅H₅N: C, 67.10; H, 5.63; N, 9.39. Found: C,67.11; H, 5.70; N, 9.42.

Hydrochloric acid solution (20 mL of 1.0 N) was added to a stirredsolution of the above salt (2.94 g) in tetrahydrofuran (25 mL). Water(100 mL) was added and the precipitate of product was filtered off anddried to give 2.38 g of the title compound, mp 160–163° C. The bulk ofthe product was recrystallized from ethyl acetate to give 2.08 g, mp160–162° C. HPLC Rt 5.85 min. ¹H NMR (CDCl₃) δ 2.32 (s, 3H), 2.37 (s,3H), 2.66 (s, 4H), 6.01 (s, 3H), 7.05–7.25 (m, 3H), 7.40 (s, 1H), 7.49(dd, 1H), and 13.0 s, 1H). ¹H NMR [(CD₃)₂SO] δ 2.32 (s, 3H), 2.35 (s,3H), 2.52 (m, 4H), 5.90 (s, 2H), 6.06 (d, 1H), 7.05–7.25 (m, 3H), 7.64(s, 1H), 7.80 (dd, 1H), 12.2 (s, 1 H), and 13.1 (s, 1H). Anal. Calcd forC₂₀H₂₀N₂O₅: C, 65.21; H, 5.47; N, 7.60. Found: C, 65.14; H, 5.49; N,7.60.

Example 4 Synthesis of1-{(3Z)-3-[(3,5-dimethyl-1H-pyrrol-2-yl)-methylidene]-2-oxo-2,3-dihydro-1H-indol-1-yl}ethylacetate (4a)

A mixture of acetic anhydride (2.1 g, 20 mmol), acetaldehyde (4.4 g, 100mmol), triethylamine (2.0 g, 20 mmol) and3-(3,5-dimethyl-1H-pyrrol-2-ylmethylidene)-1,3-dihydro-indol-2-one (2.38g, 10 mmol) in DMF (30 mL) was stirred at room temperature for 3 days.The solvents were removed and the residual oil was chromatographed onsilica gel to give, as the first product eluted from the column, 2.5 gof product, which was crystallized from ether to give 2.1 g of the titlecompound, mp 107–110° C. HPLC Rt 6.78 min. ¹H NMR (CDCl₃) δ 1.81 (d,3H), 2.09 (s, 3H), 2.33 (s, 3H), 2.38 (s, 3H), 5.98 (s, 2H), 7.00–7.20(m, 3H), 7.25 (dd, 1H), 7.39 (s, 1H), 7.51 (dd, 1H) and 13.0 (br s, 1H).Anal. Calcd for C₁₉H₂₀N₂O₃: C, 70.35; H, 6.21; N, 8.64. Found: C, 70.33;H, 6.18; N, 8.60.

Continued elution of the column gave 0.2 g of(3Z)-3-[(3,5-dimethyl-1H-pyrrol-2-yl)methylidene]-1-(1-hydroxyethyl)-1,3-dihydro-2H-indol-2-one(4b), mp 145 (dec). ¹H NMR (CDCl₃) δ 1.76 (d, 3H), 2.33 (s, 3H), 2.38(s, 3H), 3.71 (br s, 1H), 5.98 (d, 2H), 6.15 (q, 1H), 7.00–7.25 (m, 3H),7.34 (s, 1H), 7.47 (dd, 1H) and 13.0 (br s, 1H). Anal. Calcd forC₁₇H₁₈N₂O₂: C, 72.32; H, 6.43; N, 9.92. Found: C, 72.22; H, 6.52; N,9.79.

Alternatively,(3Z)-3-[(3,5-dimethyl-1H-pyrrol-2-yl)methylidene]-1-(1-hydroxyethyl)-1,3-dihydro-2H-indol-2-one(4b) was prepared in >90% yield by treating1-{(3Z)-3-[(3,5-dimethyl-1H-pyrrol-2-yl)-methylidene]-2-oxo-2,3-dihydro-1H-indol-1-yl}ethylacetate (4a) (500 mg) with aqueous acetonitrile (10 mL) and formic acid(200 mg) overnight.

Example 5 Synthesis of3-[5-{(Z)-[1-(hydroxymethyl)-1,2-dihydro-3H-indol-3-ylidine]methyl}-2,4-dimethyl-1H-pyrrole-3-propanoicacid (5)

Aqueous formaldehyde (500 mg of 38% solution, 5 mmol) was added to astirred solution of3-[5-{(Z)-[1,2-dihydro-3H-indol-3-ylidine]methyl}-2,4-dimethyl-1H-pyrrole-3-propanoicacid (310 mg, 1 mmol) and triethylamine (200 mg, 2 mmol) indimethylformamide (3 mL). After 1 h, the solution was diluted with 1 Nhydrochloric acid (5 mL) and the precipitate was filtered off, washedwith water, and dried to give 210 mg of the title compound. HPLC Rt 5.91min. ¹H NMR [(CD₃)₂SO] δ 2.27 (s, 3H), 2.32 (s, 3H), 2.36 (t, 2), 2.65(t,2H), 5.25 (d, 2), 6.26 (t, 1H), 7.05 (m, 1H), 7.15 (m, 2H), 7.62 (s,1H), 7.79 (d, 1H), 12.0 (br s, 1H) and 13.3 (br s, 1H).

Example 6 Synthesis of dibenzyl{(3Z)-3-[(3,5-dimethyl-1H-pyrrol-2-yl)methylidene]-2-oxo-2,3-dihydro-1H-indol-1-yl}methylphosphate (6)

The title compound was prepared from(3Z)-3-[(3,5-dimethyl-1H-pyrrol-2-yl)-methylidene]-1-(hydroxymethyl)-1,3-dihydro-2H-indol-2-one(1) and dibenzyl phosphorochloridate (modification of procedure used toprepare{3(Z)-3-[(3,5-dimethyl-1H-pyrrol-2-yl)-methylidene]-2-oxo-2,3-dihydro-1H-indol-1-yl}methylacetate (2)).

Example 7 Synthesis of{(3Z)-3-[(3,5-dimethyl-1H-pyrrol-2-yl)methylidene]-2-oxo-2,3-dihydro-1H-indol-1-yl}methyldihydrogen phosphate (7)

The title compound was prepared by hydrogenation of dibenzyl{(3Z)-3-[(3,5-dimethyl-1H-pyrrol-2-yl)methylidene]-2-oxo-2,3-dihydro-1H-indol-1-yl}methylphosphate (6) (modification of procedure used to prepare(3Z)-1-(aminoacetyl)-3-{[3,5-dimethyl-4-(2-carboxyethyl)-1H-pyrrol-2-yl]-methylidene}-1,3-dihydro-2H-indol-2-one(20) vide infra).

Example 8 Synthesis of benzyl{(3Z)-3-[(3,5-dimethyl-1H-pyrrol-2-yl)methylidene]-2-oxo-2,3-dihydro-1H-indol-1-yl}methylmethyl phosphate (8)

The title compound was prepared from(3Z)-3-[(3,5-Dimethyl-1H-pyrrol-2-yl)-methylidene]-1-(hydroxymethyl)-1,3-dihydro-2H-indol-2-one(1) and benzyl methyl phosphorochloridate (modification of procedureused to prepare{3(Z)-3-[(3,5-dimethyl-1H-pyrrol-2-yl)-methylidene]-2-oxo-2,3-dihydro-1H-indol-1-yl}methylacetate (2)).

Example 9 Synthesis of{(3Z)-3-[(3,5-dimethyl-1H-pyrrol-2-yl)methylidene]-2-oxo-2,3-dihydro-1H-indol-1-yl}methylmethyl hydrogen phosphate (9)

The title compoundd was prepared by hydrogenation of benzyl{(3Z)-3-[(3,5-dimethyl-1H-pyrrol-2-yl)methylidene]-2-oxo-2,3-dihydro-1H-indol-1-yl}methylmethyl phosphate (8) (modification of procedure used to prepare(3Z)-1-(aminoacetyl)-3-{[3,5-dimethyl-4-(2-carboxyethyl)-1H-pyrrol-2-yl]-methylidene}-1,3-dihydro-2H-indol-2-one(20) vide infra).

Example 10 Synthesis of{(3Z)-3-[(3,5-dimethyl-1H-pyrrol-2-yl)methylidene]-2-oxo-2,3-dihydro-1H-indol-1-yl}methyl(dimethylamino)acetate (10)

Following the procedure of Example 2, the title compound was preparedfrom 2-chloro-N,N-dimethyl-2-oxoethanaminium chloride and(3Z)-3-[(3,5-dimethyl-1H-pyrrol-2-yl)-methylidene]-1-(hydroxymethyl)-1,3-dihydro-2H-indol-2-one(1).

Example 11 Synthesis of2-({(3Z)-3-[(3,5-dimethyl-1H-pyrrol-2-yl)methylidene]-2-oxo-2,3-dihydro-1H-indol-1-yl}methoxy)-N,N,N-trimethyl-2-oxoethanaminiumchloride (11)

Following the procedure of Example 2, the title compound was preparedfrom 2-chloro-N,N,N-trimethyl-2-oxoethanaminium chloride and(3Z)-3-[(3,5-dimethyl-1H-pyrrol-2-yl)-methylidene]-1-(hydroxymethyl)-1,3-dihydro-2H-indol-2-one(1).

B. Aminomethyl Substituted Indolinones (III)

Example 12 Synthesis of1-({(3Z)-3-[(3,5-dimethyl-1H-pyrrol-2-yl)-methylidene]-2-oxo-1,3-dihydro-1H-indol-1-yl}methyl)pyridiniumchloride (12)

Phosphorus oxychloride (3.1 g, 10 mmol) was added at 0° C. to a stirredsolution of(3Z)-3-[(3,5-dimethyl-1H-pyrrol-2-yl)-methylidene]-1-(hydroxymethyl)-1,3-dihydro-2H-indol-2-one(1). (2.68 g, 10 mmol) in pyridine (20 mL). After 130 min, the solutionwas diluted slowly with water (20 mL) and the precipitate was filteredoff, washed with water, and dried to give 3.3 g of the title compound,mp >280° C. HPLC Rt 4.78 min. ¹H NMR [(CD₃OD] δ 2.35 (s, 3H), 2.37 (s,3H), 6.07 (d, 1H), 6.71 (s, 2H), 7.12–7.32 (m, 3H), 7.62 (s, 1H), 7.68(dd, 1H), 8.17, (m, 2H), 8.67 (m, 1H), 9.3 (d, 1H), and 13.1 (br s, 1H).Anal. Calcd for C₂₁H₂₀ClN₃O: C, 68.94; H, 5.51; Cl, 9.69; N, 11.48.Found: C, 68.63; H, 5.53; Cl, 9.53; N, 11.45.

Example 13 Synthesis of(3Z)-3-[(3,5-dimethyl-1H-pyrrol-2-yl)-methylidene]-1-[1-(4-methylpiperazinyl)methyl]-1,3-dihydro-2H-indol-2-one(13)

N-Methylpiperazine (10 g, 100 mmol) was added to a stirred solution ofaqueous formaldehyde (10 g of 38% solution, 100 mmol) and3-(3,5-dimethyl-1H-pyrrol-2-ylmethylidene)-1,3-dihydro-indol-2-one,(2.38 g, 10 mmol) in methanol (100 mL). The solution heated at 60° C.for 1 h, concentrated to a low volume and the precipitate was filteredoff, washed with methanol, and dried to give 2.38 g of the titlecompound, mp 160–164° C. HPLC Rt 4.72 min. ¹H NMR (CDCl₃) δ 2.26 (s,3H), 2.33 (s, 3H), 2.38 (s, 3H), 2.43 (br s, 4H), 2.70 (br s, 4H), 4.59(s, 2H), 5.96 (d, 1H), 7.02–7.08 (m, 2H), 7.15 (dd, 1H), 7.38 (s, 1H),7.48 (dd, 1H) and 13.0 (br s, 1H). Anal. Calcd for C₂₁H₂₆N₄O: C, 71.97;H, 7.48; N, 15.99. Found: C, 71.75; H, 7.46; N, 15.87.

(3Z)-3-[(3,5-Dimethyl-1H-pyrrol-2-yl)-methylidene]-1-[1-(4-methylpiperazinyl)-methyl]-1,3-dihydro-2H-indol-2-one(13) has been converted to a dihydrochloride salt.

Example 14 Synthesis of(3Z)-3-[(3,5-dimethyl-1H-pyrrol-2-yl)-methylidene]-1-(1-pyrrolidinylmethyl)-1,3-dihydro-2H-indol-2-one(14)

Pyrrolidine (450 mg, 6.3 mmol) was added to a stirred solution ofaqueous formaldehyde (500 mg of 38% solution, 6.0 mmol) and3-(3,5-dimethyl-1H-pyrrol-2-ylmethylidene)-1,3-dihydro-indol-2-one, (900mg, 3.8 mmol) in methanol (50 mL). After 15 min, the solution was cooledto 0° C. and the precipitate was filtered off, washed with water, anddried to give 1.08 g of the title compound, mp 129–132° C. HPLC Rt 4.87min. ¹H NMR [(CD₃)₂SO] δ 1.65 (m, 4H), 2.32 9s, 3H), 2.34 (s, 3H), 2.62(m, 4H), 4.72 (s, 2H) 6.07 (d, 1H), 7.00 (m, 1H), 7.15 (m, 2H), 7.61 (s,1H), 7.76 (d, 2H) and 13.1 (br s, 1H). Anal. Calcd for C₂₀H₂₃N₃O: C,74.74; H, 7.21; N, 13.07. Found: C, 74.61; H, 7.25; N, 13.03.

C. Acyl Substituted Indolinones (III)

Example 15 Synthesis of(3Z)-1-Acetyl-3-[(3,5-dimethyl-4-1H-pyrrol-2-yl)-methylidene]-1,3-dihydro-2H-indol-2-one(15)

The title compound was prepared according to Procedure 1 or Procedure 2.

Procedure 1: Sodium hydride (200 mg of 60% in oil) was added to astirred solution of3-(3,5-dimethyl-1H-pyrrol-2-ylmethylidene)-1,3-dihydro-indol-2-one (1.19g, 5 mmol) in DMF (30 mL). After 10 min, acetic anhydride (1.02 g, 10mmol) in DMF (10 mL) was added. Water (80 mL) was added to completeprecipitation of the product. The dried product was chromatographed onsilica gel with chloroform as the eluant to give 1.05 g of the titlecompound which was triturated with ether; mp 193–196° C. HPLC Rt 6.98min. ¹H NMR (CDCl₃) δ 2.34 (s, 3H), 2.42 (s, 3H), 2.79 (s, 3H), 6.06 (d,1H), 7.19 (m, 2H), 7.38 (s, 1H), 7.42 (m, 1H), 8.24 (m, 1H) and 12.6 (brs, 1H). Anal. Calcd for C₁₇H₁₆N₂O₂: C, 72.84; H, 5.75; N, 9.99. Found:C, 72.55; H, 5.50; N, 9.86.

Procedure 2: A mixture of3-(3,5-dimethyl-1H-pyrrol-2-ylmethylidene)-1,3-dihydro-indol-2-one (930mg, 3.9 mmol) and acetic anhydride (15 mL, excess) was heated at 95° C.for several days. TLC showed no SM. The reaction was cooled to roomtemperature and the resulting precipitate was collected by vacuumfiltration, washed with water and dried to give 1.03 g (94%) of thetitle compound. ¹HNMR (300 MHz, DMSO-d6) δ 12.54 (v br s, 1H, NH), 8.10(m, 1H), 7.85 (m, 1H), 7.64 (s, 1H, H-vinyl), 7.2 (m, 2H), 6.12 (d,J=2.4 Hz, 1H), 2.70 (s, 3H, COCH₃), 2.38 (s, 3H, CH₃), 2.34 (s, 3H,CH₃). MS MH⁺ 281.2.

Example 16 Synthesis of(3Z)-1-[(Dimethylamino)acetyl)-3-[(3,5-dimethyl-4-1H-pyrrol-2-yl)-methylidene]-1,3-dihydro-2H-indol-2-one(16)

The title compound was prepared from3-(3,5-dimethyl-1H-pyrrol-2-ylmethylidene)-1,3-dihydro-indol-2-one and2-chloro-N,N-dimethyl-2-oxoethanaminium chloride (modification ofprocedure used to prepare(3Z)-1-acetyl-3-[(3,5-dimethyl-4-1H-pyrrol-2-yl)-methylidene]-1,3-dihydro-2H-indol-2-one(15)).

Example 17 Synthesis of2-{(3Z)-3-[(3,5-dimethyl-1H-pyrrol-2-yl)-methylidene]-2-oxo-2,3-dihydro-1H-indol-1-yl}-N,N,N-trimethyl-2-oxo-1-ethanaminiumchloride (17)

The title compound was prepared from3-(3,5-dimethyl-1H-pyrrol-2-ylmethylidene)-1,3-dihydro-indol-2-one and2-chloro-N,N,N-trimethyl-2-oxoethanaminium chloride (modification ofprocedure used to prepare(3Z)-1-acetyl-3-[(3,5-dimethyl-4-1H-pyrrol-2-yl)-methylidene]-1,3-dihydro-2H-indol-2-one(15)).

Example 18 Synthesis of4-{(3Z)-3-[(3,5-dimethyl-1H-pyrrol-2-yl)-methylidene]-2-oxo-2,3-dihydro-1H-indol-1-yl}-4-oxobutanoicacid (18)

The title compound was prepared from3-(3,5-dimethyl-1H-pyrrol-2-ylmethylidene)-1,3-dihydro-indol-2-one andsuccinic anhydride (modification of procedure used to prepare(3Z)-1-acetyl-3-[(3,5-dimethyl-4-1H-pyrrol-2-yl)-methylidene]-1,3-dihydro-2H-indol-2-one(15)).

Example 19 Synthesis of benzyl2-{(3Z)-3-[(3,5-dimethyl-1H-pyrrol-2-yl)methylidene]-2-oxo-2,3-dihydro-1H-indol-1-yl}-2-oxoethylcarbamate(19)

A mixture of N-(benzyloxycarbonyl)glycine (4.9 g, 23 mmol),3-(3,5-dimethyl-1H-pyrrol-2-ylmethylidene)-1,3-dihydro-indol-2-one (2.38g, 10 mmol), dimethylaminopyridine (1.28 g, 10 mmol) in DMF (20 mL) washeated at 55° C. for 2 h. The solution was cooled, ether (10 mL) wasadded and the precipitate of the title compound was filtered off anddried to give 2.35 g, mp 193–196° C. HPLC Rt 7.04 min. ¹H NMR (CDCl₃) δ2.34 (s, 3H), 2.42 (s, 3H), 4.87 (d, 2H), 5.18 (s, 2H), 5.74 (t, 1H),6.06 (d, 1H), 7.2 (m, 3H), 7.3-7-65 (m, 7H), 7.49 (m, 1H), and 8.23 (m,1H). Anal. Calcd for C₂₅H₂₃N₃O₄: C, 69.92; H, 5.40; N, 9.78. Found: C,69.91; H, 5.50; N, 9.86.

Example 20 Synthesis of(3Z)-1-(aminoacetyl)-3-{[3,5-dimethyl-4-(2-carboxyethyl)-1H-pyrrol-2-yl]-methylidene}-1,3-dihydro-2H-indol-2-one(20)

Benzyl2-{(3Z)-3-[(3,5-dimethyl-1H-pyrrol-2-yl)methylidene]-2-oxo-2,3-dihydro-1H-indol-1-yl}-2-oxoethylcarbamate(19) (160 mg) was dissolved in ethyl acetate (70 mL). 10% Falladium oncarbon (200 mg) was added and the mixture was hydrogenated at 50 psihydrogen pressure for 1 h to give the title compound in 35% yield; HPLCRt 4.63 min.

Example 21 Synthesis of(3Z)-3-[(3,5-dimethyl-1H-pyrrol-2-yl)-methylidene]-1-(dimethylphosphoryl)-1,3-dihydro-2H-indol-2-one(21)

(3Z)-3-[(3,5-dimethyl-1H-pyrrol-2-yl)-methylidene]-1,3-dihydro-2H-indol-2-one(521 mg, 2.19 mmol) was dissolved in THF (60 ml). The reaction mixturewas cooled to −78° C. Butyllithium (2.7 ml, 4.32 mmol; 1.6 M in hexane)was added dropwise followed by the dropwise addition of dimethylchlorophosphate (0.46 ml, 4.27 mmol). The reaction mixture was stirredfor 1 h at −78° C., then the mixture was allowed to warm to 0° C. during4 h. The reaction mixture was poured into -ice water and extracted withEtOAc (2×). The organic layers were washed with brine and dried oversodium sulfate. The solvent was removed and the residue was purified bysilica gel chromatography (CH₂Cl₂/EtOAc: 10/1; 250 ml; CH₂Cl₂/EtOAc:3/1; 300 ml) to yield the title compound as a red solid (87%).

¹H NMR (400 MHz, d₆-DMSO) δ 2.35 (s, 3H), 2.38 (s, 3H), 3.80 (d, J=12.1Hz, 6H), 6.12 (d, J=2.3 Hz, 1H), 7.13–7.20 (m, 2H), 7.67 (d, J=1.2 Hz,1H), 7.71 (dd, J=2.0, 7.2 Hz, 1H), 7.84 (dd, J=2.0, 7.0 Hz, 1H), 12.64(s, 1H); ³¹P NMR (162 MHz, d₆-DMSO) δ −1.05.

Example 22 Synthesis of(3Z)-3-[(3,5-dimethyl-1H-pyrrol-2-yl)-methylidene]-1-(phosphoryl)-1,3-dihydro-2H-indol-2-one(22)

(3Z)-3-[(3,5-Dimethyl-1H-pyrrol-2-yl)-methylidene]-1-(dimethylphosphoryl)-1,3-dihydro-2H-indol-2-one(648 mg, 1.87 mmol) was dissolved in CH₃CN (11 ml).N,O-bis(trimethylsilyl)acetamide (0.53 ml, 2.14 mmol) andtrimetylsilylbromide (0.53 ml, 4.02 mmol) were added dropwise at rt. Thedark red solution was stirred for 14 h at rt. The solution wasdistributed into two scintillation vials and water (0.2 ml per vial) wasadded. The product precipitated out. The vials were centrifuged and thesolvent was decanted. The orange/red solid was washed with EtOAc (3×) toprovide the title compound (94%).

¹H NMR (400 MHz, d₆-DMSO) δ 2.33 (s, 3H), 2.35 (s, 3H), 6.08 (d, J=2.0Hz, 1H), 7.05–7.15 (m, 2H), 7.61 (s, 1H), 7.78 (d, J=7.8 Hz, 1H), 7.83(d, J=7.8 Hz, 1H), 12.99 (s, 1H); ³¹P NMR (162 MHz, d₆-DMSO) δ −6.98.

Biological Evaluation

It will be appreciated that, in any given series of compounds, a rangeof biological activities will be observed. In its presently preferredembodiments, this invention relates to novel1-susbtituted-3-pyrrolidinyl-2-indolinones capable of generating in vivo3-pyrrolidinyl-2-indolinones capable of modulating, regulating and/orinhibiting protein kinase activity. The following assays may be employedto select those compounds demonstrating the optimal degree of thedesired activity.

Assay Procedures

The following in vitro assays may be used to determine the level ofactivity and effect of the different compounds of the present inventionon one or more of the PKs. Similar assays can be designed along the samelines for any PK using techniques well known in the art.

Several of the assays described herein are performed in an ELISA(Enzyme-Linked Immunosorbent Sandwich Assay) format (Voller, et al.,1980, “Enzyme-Linked Immunosorbent Assay,” Manual of ClinicalImmunology, 2d ed., Rose and Friedman, Am. Soc. Of Microbiology,Washington, D.C., pp. 359–371). The general procedure is as follows: acompound is introduced to cells expressing the test kinase, either.naturally or recombinantly, for a selected period of time after which,if the test kinase is a receptor, a ligand known to activate thereceptor is added. The cells are lysed and the lysate is transferred tothe wells of an ELISA plate previously coated with a specific antibodyrecognizing the substrate of the enzymatic phosphorylation reaction.Non-substrate components of the cell lysate are washed away and theamount of phosphorylation on the substrate is detected with an antibodyspecifically recognizing phosphotyrosine compared with control cellsthat were not contacted with a test compound.

The presently preferred protocols for conducting the ELISA experimentsfor specific PKs is provided below. However, adaptation of theseprotocols for determining the activity of compounds against other RTKs,as well as for CTKs and STKs, is well within the scope of knowledge ofthose skilled in the art. Other assays described herein measure theamount of DNA made in response to activation of a test kinase, which isa general measure of a proliferative response. The general procedure forthis assay is as follows: a compound is introduced to cells expressingthe test kinase, either naturally or recombinantly, for a selectedperiod of time after which, if the test kinase is a receptor, a ligandknown to activate the receptor is added. After incubation at leastovernight, a DNA labeling reagent such as 5-bromodeoxyuridine (BrdU) orH³-thymidine is added. The amount of labeled DNA is detected with eitheran anti-BrdU antibody or by measuring radioactivity and is compared tocontrol cells not contacted with a test compound.

GST-Flk-1 Bioassay

This assay analyzes the tyrosine kinase activity of GST-Flk1 onpoly(glu-tyr) peptides.

Materials and Reagents:

-   -   1. Corning 96-well ELISA plates (Corning Catalog No. 25805-96).    -   2. poly(glu-tyr) 4:1, lyophilizate (Sigma Catalog No. P0275), 1        mg/ml in sterile PBS.    -   3. PBS Buffer: for 1 L, mix 0.2 g KH₂PO₄, 1.15 g Na₂HPO₄, 0.2 g        KCl and 8 g NaCl in approx. 900 ml dH₂O. When all reagents have        dissolved, adjust the pH to 7.2 with HCl. Bring total volume to        1 L with dH₂O.    -   4. PBST Buffer: to 1 L of PBS Buffer, add 1.0 ml Tween-20.    -   5. TBB—Blocking Buffer: for 1 L, mix 1.21 g TRIS, 8.77 g NaCl, 1        ml TWEEN-20 in approximately 900 ml dH₂O. Adjust pH to 7.2 with        HCl. Add 10 g BSA, stir to dissolve. Bring total volume to 1 L        with dH₂O. Filter to remove particulate matter.    -   6. 1% BSA in PBS: add 10 g BSA to approx. 990 ml PBS buffer,        stir to dissolve. Adjust total volume to 1 L with PBS buffer,        filter to remove particulate matter.    -   7. 50 mM Hepes pH 7.5.    -   8. GST-Flk1cd purified from sf9 recombinant baculovirus        transformation (SUGEN, Inc.).    -   9. 4% DMSO in dH₂O.    -   10. 10 mM ATP in dH₂O.    -   11. 40 mM MnCl₂    -   12. Kinase Dilution Buffer (KDB): mix 10 ml Hepes (pH 7.5), 1 ml        5M NaCl, 40 μL 100 mM sodium orthovanadate and 0.4 ml of 5% BSA        in dH₂O with 88.56 ml dH₂O.    -   13. NUNC 96-well V bottom polypropylene plates, Applied        Scientific Catalog # AS-72092    -   14. EDTA: mix 14.12 g ethylenediaminetetraacetic acid (EDTA)        with approx. 70 ml dH₂O. Add 10 N NaOH until EDTA dissolves.        Adjust pH to 8.0. Adjust total volume to 100 ml with dH₂O.    -   15. 1° and 2° Antibody Dilution Buffer: mix 10 ml of 5% BSA in        PBS buffer with 89.5 ml TBST.    -   16. Anti-phosphotyrosine rabbit polyclonal antisera (SUGEN,        Inc.)    -   17. Goat anti-rabbit HRP conjugate.    -   18. ABST solution: To approx. 900 ml dH₂O add 19.21 g citric        acid and 35.49 g Na₂HPO₄. Adjust pH to 4.0 with phosphoric acid.        Add 2,2′-Azinobis(3-ethyl-benzthiazoline-6-sulfonic acid (ABTS,        Sigma, Cat. No. A-1888, hold for approx. ½ hour, filter.    -   19. 30% Hydrogen Peroxide.    -   20. ABST/H₂O₂: add 3 μl of H₂O₂ to 15 ml of ABST solution.    -   21. 0.2 M HCl.        Procedure:    -   1. Coat Corning 96-well ELISA plates with 2 μg of polyEY in 100        μl PBS/well, hold at room temperature for 2 hours or at 4° C.        overnight. Cover plates to prevent evaporation.    -   2. Remove unbound liquid from wells by inverting plate. Wash        once with TBST. Pat the plate on a paper towel to remove excess        liquid.    -   3. Add 100 μl of 1% BSA in PBS to each well. Incubate, with        shaking, for 1 hr. at room temperature.    -   4. Repeat step 2.    -   5. Soak wells with 50 mM HEPES (pH7.5, 150 μl/well).    -   6. Dilute test compound with dH₂O/4% DMSO to 4 times the desired        final assay concentration in 96-well polypropylene plates.    -   7. Add 25 μl diluted test compound to each well of ELISA plate.        In control wells, place 25 μl of dH₂O/4% DMSO.    -   8. Dilute GST-Flk1 0.005 μg (5 ng)/well in KDB.    -   9. Add 50 μl of diluted enzyme to each well.    -   10. Add 25 μl 0.5 M EDTA to negative control wells.    -   11. Add 25 μl of 40 mM MnCl, with 4×ATP (2 μM) to all wells (100        μl final volume, 0.5 μM ATP final concentration in each well).    -   12. Incubate, with shaking, for 15 minutes at room temperature.    -   13. Stop reaction by adding 25 μl of 500 mM EDTA to each well.    -   14. Wash 3× with TBST and pat plate on paper towel to remove        excess liquid.    -   15. Add 100 μl per well anti-phosphotyrosine antisera, 1:10,000        dilution in antibody dilution buffer. Incubate, with shaking,        for 90 min. at room temperature.    -   16. Wash as in step 14.    -   17. Add 100 μl/well of goat anti-rabbit HRP conjugate (1:6,000        in antibody dilution buffer). Incubate, with shaking, for 90        minutes are room temperature.    -   18. Wash as in Step 14.    -   19. Add 100 μl room temperature ABST/H₂O₂ solution to each well.    -   20. Incubate, with shaking for 15 to 30 minutes at room        temperature.    -   21. If necessary, stop reaction by adding 100 μl of 0.2 M HCl to        each well.    -   22. Read results on Dynatech MR7000 ELISA reader with test        filter at 410 nM and reference filter at 630 nM.        PYK2 Bioassay    -   This assay is used to measure the in vitro kinase activity of HA        epitope-tagged full length pyk2 (FL.pyk2-HA) in an ELISA assay.        Materials and Reagents:    -   1. Corning 96-well ELISA plates.    -   2. 12CA5 monoclonal anti-HA antibody (SUGEN, Inc.)    -   3. PBS (Dulbecco's Phosphate-Buffered Saline (Gibco Catalog #        450-1300EB)    -   4. TBST Buffer: for 1 L, mix 8.766 g NaCl, 6.057 g TRIS and 1 ml        of 0.1% Triton X-100 in approx. 900 ml dH₂O. Adjust pH to 7.2,        bring volume to 1 L.    -   5. Blocking Buffer: for 1 L, mix 100 g 10% BSA, 12.1 g 100 mM        TRIS, 58.44 g 1M NaCl and 10 mL of 1% TWEEN-20.    -   6. FL.pyk2-HA from sf9 cell lysates (SUGEN, Inc.).    -   7. 4% DMSO in MilliQue H₂O.    -   8. 10 mM ATP in dH₂O.    -   9. 1M MnCl₂.    -   10 1M MgCl₂.    -   11. 1M Dithiothreitol (DTT).    -   12. 10× Kinase buffer phosphorylation: mix 5.0 ml 1M Hepes (pH        7.5), 0.2 ml 1M MnCl₂, 1.0 ml 1 M MgCl₂, 1.0 ml 10% Triton X-100        in 2.8 ml dH₂O. Just prior to use, add 0.1 ml 1M DTT.    -   13. NUNC 96-well V bottom polypropylene plates.    -   14. 500 mM EDTA in dH₂O.    -   15. Antibody dilution buffer: for 100 mL, 1 mL 5% BSA/PBS and 1        mL 10% Tween-20 in 88 mL TBS.    -   16. HRP-conjugated anti-Ptyr (PY99, Santa Cruz Biotech Cat. No.        SC-7020).    -   17. ABTS, Moss, Cat. No. ABST-2000.    -   18. 10% SDS.        Procedure:    -   1. Coat Corning 96 well ELISA plates with 0.5 μg per well 12CA5        anti-HA antibody in 100 μl PBS. Store overnight at 4° C.    -   2. Remove unbound HA antibody from wells by inverting plate.        Wash plate with dH₂O. Pat the plate on a paper towel to remove        excess liquid.    -   3. Add 150 μl Blocking Buffer to each well. Incubate, with        shaking, for 30 min at room temperature.    -   4. Wash plate 4× with TBS-T.    -   5. Dilute lysate in PBS (1.5 μg lysate/100 μl PBS).    -   6. Add 100 μl of diluted lysate to each well. Shake at room        temperature for 1 hr.    -   7. Wash as in step 4.    -   8. Add 50 μl of 2× kinase Buffer to ELISA plate containing        captured pyk2-HA.    -   9. Add 25 μL of 400 μM test compound in 4% DMSO to each well.        For control wells use 4% DMSO alone.    -   10. Add 25 μL of 0.5 M EDTA to negative control wells.    -   11. Add 25 μl of 20 μM ATP to all wells. Incubate, with shaking,        for 10 minutes.    -   12. Stop reaction by adding 25 μl 500 mM EDTA (pH 8.0) to all        wells.    -   13. Wash as in step 4.    -   14. Add 100 μL HRP conjugated anti-Ptyr diluted 1:6000 in        Antibody Dilution Buffer to each well. Incubate, with shaking,        for 1 hr. at room temperature.    -   15. Wash plate 3× with TBST and 1× with PBS.    -   16. Add 100 μL of ABST solution to each well.    -   17. If necessary, stop the development reaction by adding 20 μL        10% SDS to each well.    -   18. Read plate on ELISA reader with test filter at 410 nM and        reference filter at 630 nM.        FGFR1 Bioassay

This assay is used to measure the in vitro kinase activity of FGF1-R inan ELISA assay.

Materials and Reagents:

-   -   1. Costar 96-well ELISA plates (Corning Catalog # 3369).    -   2. Poly(Glu-Tyr) (Sigma Catalog # PO275).    -   3. PBS (Gibco Catalog # 450-1300EB)    -   4. 50 mM Hepes Buffer Solution.    -   5. Blocking Buffer (5% BSA/PBS).    -   6. Purified GST-FGFR1 (SUGEN, Inc.)    -   7. Kinase Dilution Buffer. Mix 500 μl 1M Hepes (GIBCO), 20 μl 5%        BSA/PBS, 10 μl 100 mM sodium orthovanadate and 50 μl 5M NaCl.    -   8. 10 mM ATP    -   9. ATP/MnCl₂ phosphorylation mix: mix 20 μL ATP, 400 μL 1M MnCl₂        and 9.56 ml dH₂O.    -   10. NUNC 96-well V bottom polypropylene plates (Applied        scientific Catalog # AS-72092).    -   11. 0.5M EDTA.    -   12. 0.05% TBST Add 500 μL TWEEN to 1 liter TBS.    -   13. Rabbit polyclonal anti-phosphotyrosine serum (SUGEN, Inc.).    -   14. Goat anti-rabbit IgG peroxidase conjugate (Biosource,        Catalog # ALI0404).    -   15. ABTS Solution.    -   16. ABTS/H₂O₂ solution.        Procedure:    -   1. Coat Costar 96 well ELISA plates with 1 μg per well        Poly(Glu-Tyr) in 100 μl PBS. Store overnight at 4° C.    -   2. Wash coated plates once with PBS.    -   3. Add 150 μL of 5% BSA/PBS Blocking Buffer to each well.        Incubate, with shaking, for 1 hr at room temperature.    -   4. Wash plate 2× with PBS, then once with 50 mM Hepes. Pat        plates on a paper towel to remove excess liquid and bubbles.    -   5. Add 25 μL of 0.4 mM test compound in 4% DMSO or 4% DMSO alone        (controls) to plate.    -   6. Dilute purified GST-FGFR1 in Kinase Dilution Buffer (5 ng        kinase/50 ul KDB/well).    -   7. Add 50 μL of diluted kinase to each well.    -   8. Start kinase reaction by adding 25 μl/well of freshly        prepared ATP/Mn++ (0.4 ml 1M MnCl₂, 40 μL 10 mM ATP, 9.56 ml        dH₂O), freshly prepared).    -   9. Stop reaction with 25 μL of 0.5M EDTA.    -   10. Wash plate 4× with fresh TBST.    -   11. Make up Antibody Dilution Buffer: For 50 ml, mix 5 ml of 5%        BSA, 250 μl of 5% milk and 50 μl of 100 mM sodium vanadate,        bring to final volume with 0.05% TBST.    -   12. Add 100 μl per well of anti-phosphotyrosine (1:10000        dilution in ADB). Incubate, with shaking for 1 hr. at room        temperature.    -   13. Wash as in step 10.    -   14. Add 100 μl per well of Biosource Goat anti-rabbit IgG        peroxidase conjugate (1:6000 dilution in ADB). Incubate, with        shaking for 1 hr. at room temperature.    -   15. Wash as in step 10 and then with PBS to remove bubbles and        excess TWEEN.    -   16. Add 100 μl of ABTS/H₂O₂ solution to each well.    -   17. Incubate, with shaking, for 10 to 20 minutes. Remove any        bubbles.    -   18. Read assay on Dynatech MR7000 ELISA reader: test filter at        410 nM, reference filter at 630 nM.        EGFR Bioassay

This assay is used to the in vitro kinase activity of EGFR in an ELISAassay.

Materials and Reagents:

-   -   1. Corning 96-well ELISA plates.    -   2. SUMO1 monoclonal anti-EGFR antibody (SUGEN, Inc.).    -   3. PBS.    -   4. TBST Buffer.    -   5. Blocking Buffer: for 100 ml, mix 5.0 g Carnation® Instant        Non-fat Milk with 100 ml of PBS.    -   6. A431 cell lysate (SUGEN, Inc.).    -   7. TBS Buffer.    -   8. TBS+10% DMSO: for 1 L, mix 1.514 g TRIS, 2.192 g NaCl and 25        ml DMSO; bring to 1 liter total volume with dH₂O.    -   9. ATP (Adenosine-5′-triphosphate, from Equine muscle, Sigma        Cat. No. A-5394), 1.0 mM solution in dH₂O. This reagent should        be made up immediately prior to use and kept on ice.    -   10. 1.0 mM MnCl₂.    -   11. ATP/MnCl₂ phosphorylation mix: for 10 ml, mix 300 μl of 1 mM        ATP, 500 μl MnCl₂ and 9.2 ml dH₂O. Prepare just prior to use,        keep on ice.    -   12. NUNC 96-well V bottom polypropylene plates.    -   13. EDTA.    -   14. Rabbit polyclonal anti-phosphotyrosine serum (SUGEN, Inc.).    -   15. Goat anti-rabbit IgG peroxidase conjugate (Biosource Cat.        No. ALI0404).    -   16. ABTS.    -   17. 30% Hydrogen peroxide.    -   18. ABTS/H₂O₂    -   19. 0.2 M HCl.        Procedure:    -   1. Coat Corning 96 well ELISA plates with 0.5 μg SUMO1 in 100 μl        PBS per well, hold overnight at 4° C.    -   2. Remove unbound SUMO1 from wells by inverting plate to remove        liquid. Wash 1× with dH₂O. Pat the plate on a paper towel to        remove excess liquid.    -   3. Add 150 μl of Blocking Buffer to each well. Incubate, with        shaking, for 30 min. at room temperature.    -   4. Wash plate 3× with deionized water, then once with TBST. Pat        plate on a paper towel to remove excess liquid and bubbles.    -   5. Dilute lysate in PBS (7 μg lysate/100 μl PBS).    -   6. Add 100 μl of diluted lysate to each well. Shake at room        temperature for 1 hr.    -   7. Wash plates as in 4, above.    -   8. Add 120 μl TBS to ELISA plate containing captured EGFR.    -   9. Dilute test compound 1:10 in TBS, place in well    -   10. Add 13.5 μl diluted test compound to ELISA plate. To control        wells, add 13.5 μl TBS in 10% DMSO.    -   11. Incubate, with shaking, for 30 minutes at room temperature.    -   12. Add 15 μl phosphorylation mix to all wells except negative        control well. Final well volume should be approximately 150 μl        with 3 μM ATP/5 mM MnCl₂ final concentration in each well.        Incubate with shaking for 5 minutes.    -   13. Stop reaction by adding 16.5 μl of EDTA solution while        shaking. Shake for additional 1 min.    -   14. Wash 4× with deionized water, 2× with TBST.    -   15. Add 100 μl anti-phosphotyrosine (1:3000 dilution in TBST)        per well. Incubate, with shaking, for 30–45 min. at room        temperature.    -   16. Wash as in 4, above.    -   17. Add 100 μl Biosource Goat anti-rabbit IgG peroxidase        conjugate (1:2000 dilution in TBST) to each well. Incubate with        shaking for 30 min. at room temperature.    -   18. Wash as in 4, above.    -   19. Add 100 μl of ABTS/H₂O₂ solution to each well.    -   20. Incubate 5 to 10 minutes with shaking. Remove any bubbles.    -   21. If necessary, stop reaction by adding 100 μl 0.2 M HCl per        well.    -   22. Read assay on Dynatech MR7000 ELISA reader: test filter at        410 nM, reference filter at 630 nM.        PDGFR Bioassay

This assay is used to the in vitro kinase activity of PDGFR in an ELISAassay.

Materials and Reagents:

-   -   1. Corning 96-well ELISA plates    -   2. 28D4C10 monoclonal anti-PDGFR antibody (SUGEN, Inc.).    -   3. PBS.    -   4. TBST Buffer.    -   5. Blocking Buffer (same as for EGFR bioassay).    -   6. PDGFR-β expressing NIH 3T3 cell lysate (SUGEN, Inc.).    -   7. TBS Buffer.    -   8. TBS+10% DMSO.    -   9. ATP.    -   10. MnCl₂.    -   11. Kinase buffer phosphorylation mix: for 10 ml, mix 250 μl 1M        TRIS, 200 μl 5M NaCl, 100 μl 1M MnCl₂ and 50 μl 100 mM Triton        X-100 in enough dH₂O to make 10 ml.    -   12. NUNC 96-well V bottom polypropylene plates.    -   13. EDTA.    -   14. Rabbit polyclonal anti-phosphotyrosine serum (SUGEN, Inc.).    -   15. Goat anti-rabbit IgG peroxidase conjugate (Biosource Cat.        No. ALI0404).    -   16. ABTS.    -   17. Hydrogen peroxide, 30% solution.    -   18. ABTS/H₂O₂.    -   19. 0.2 M HCl.        Procedure:    -   1. Coat Corning 96 well ELISA plates with 0.5 μg 28D4C10 in 100        μl PBS per well, hold overnight at 4° C.    -   2. Remove unbound 28D4C10 from wells by inverting plate to        remove liquid. Wash 1× with dH₂O. Pat the plate on a paper towel        to remove excess liquid.    -   3. Add 150 μl of Blocking Buffer to each well. Incubate for 30        min. at room temperature with shaking.    -   4. Wash plate 3× with deionized water, then once with TBST. Pat        plate on a paper towel to remove excess liquid and bubbles.    -   5. Dilute lysate in HNTG (10 μg lysate/100 μl HNTG).    -   6. Add 100 μl of diluted lysate to each well. Shake at room        temperature for 60 min.    -   7. Wash plates as described in Step 4.    -   8. Add 80 μl working kinase buffer mix to ELISA plate containing        captured PDGFR.    -   9. Dilute test compound 1:10 in TBS in 96-well polypropylene        plates.    -   10. Add 10 μl diluted test compound to ELISA plate. To control        wells, add 10 μl TBS+10% DMSO. Incubate with shaking for 30        minutes at room temperature.    -   11. Add 10 μl ATP directly to all wells except negative control        well (final well volume should be approximately 100 μl with 20        μM ATP in each well.) Incubate 30 minutes with shaking.    -   12. Stop reaction by adding 10 μl of EDTA solution to each well.    -   13. Wash 4× with deionized water, twice with TBST.    -   14. Add 100 μl anti-phosphotyrosine (1:3000 dilution in TBST)        per well. Incubate with shaking for 30–45 min. at room        temperature.    -   15. Wash as in Step 4.    -   16. Add 100 μl Biosource Goat anti-rabbit IgG peroxidase        conjugate (1:2000 dilution in TBST) to each well. Incubate with        shaking for 30 min. at room temperature.    -   17. Wash as in Step 4.    -   18. Add 100 μl of ABTS/H₂O₂ solution to each well.    -   19. Incubate 10 to 30 minutes with shaking. Remove any bubbles.    -   20. If necessary stop reaction with the addition of 100 μl 0.2 M        HCl per well.    -   21. Read assay on Dynatech MR7000 ELISA reader with test filter        at 410 nM and reference filter at 630 nM.        Cellular HER-2 Kinase Assay

This assay is used to measure HER-2 kinase activity in whole cells in anELISA format.

Materials and Reagents:

-   -   1. DMEM (GIBCO Catalog #11965-092).    -   2. Fetal Bovine Serum (FBS, GIBCO Catalog #16000-044), heat        inactivated in a water bath for 30 min. at 56° C.    -   3. Trypsin (GIBCO Catalog #25200-056).    -   4. L-Glutamine (GIBCO Catalog #25030-081).    -   5.HEPES (GIBCO Catalog #15630-080).    -   6. Growth Media: Mix 500 ml DMEM, 55 ml heat inactivated FBS, 10        ml HEPES and 5.5 ml L-Glutamine.    -   7. Starve Media: Mix 500 ml DMEM, 2.5 ml heat inactivated FBS,        10 ml HEPES and 5.5 ml L-Glutamine.    -   8. PBS.    -   9. Flat Bottom 96-well Tissue Culture Micro Titer Plates        (Corning Catalog # 25860).    -   10. 15 cm Tissue Culture Dishes (Corning Catalog #08757148).    -   11. Corning 96-well ELISA Plates.    -   12. NUNC 96-well V bottom polypropylene plates.    -   13. Costar Transfer Cartridges for the Transtar 96 (Costar        Catalog #7610).    -   14. SUMO 1: monoclonal anti-EGFR antibody (SUGEN, Inc.).    -   15. TBST Buffer.    -   16. Blocking Buffer: 5% Carnation Instant Milk® in PBS.    -   17. EGF Ligand: EGF-201, Shinko American, Japan. Suspend powder        in 100 μL of 10 mM HCl. Add 100 uL 10 mM NaOH. Add 800 μL PBS        and transfer to an Eppendorf tube, store at −20° C. until ready        to use.    -   18. HNTG Lysis Buffer: For Stock 5×HNTG, mix 23.83 g Hepes,        43.83 g NaCl, 500 ml glycerol and 100 ml Triton X-100 and enough        dH₂O to make 1 L of total solution.        -   For 1×HNTG*, mix 2 ml 5×HNTG, 100 μL 0.1M Na₃VO₄, 250 μL            0.2M Na₄P₂O₇ and 100 μL EDTA.    -   19. EDTA.    -   20. Na₃VO₄: To make stock solution, mix 1.84 g Na₃VO₄ with 90 ml        dH₂O. Adjust pH to 10. Boil in microwave for one minute        (solution becomes clear). Cool to room temperature. Adjust pH        to 10. Repeat heating/cooling cycle until pH remains at 10.    -   21. 200 mM Na₄P₂O₇.    -   22. Rabbit polyclonal antiserum specific for phosphotyrosine        (anti-Ptyr antibody, SUGEN, Inc.).    -   23. Affinity purified antiserum, goat anti-rabbit IgG antibody,        peroxidase conjugate (Biosource Cat # ALI0404).    -   24. ABTS Solution.    -   25. 30% Hydrogen peroxide solution.    -   26. ABTS/H₂O₂.    -   27. 0.2 M HCl.        Procedure:    -   1. Coat Corning 96 well ELISA plates with SUMO1 at 1.0 μg per        well in PBS, 100 μl final volume/well. Store overnight at 4° C.    -   2. On day of use, remove coating buffer and wash plate 3 times        with dH₂O and once with TBST buffer. All washes in this assay        should be done in this manner, unless otherwise specified.    -   3. Add 100 μL of Blocking Buffer to each well. Incubate plate,        with shaking, for 30 min. at room temperature. Just prior to        use, wash plate.    -   4. Use EGFr/HER-2 chimera/3T3-C7 cell line for this assay.    -   5. Choose dishes having 80–90% confluence. Collect cells by        trypsinization and centrifuge at 1000 rpm at room temperature        for 5 min.    -   6. Resuspend cells in starve medium and count with trypan blue.        Viability above 90% is required. Seed cells in starve medium at        a density of 2,500 cells per well, 90 μL per well, in a 96 well        microtiter plate. Incubate seeded cells overnight at 37° under        5% CO₂.    -   7. Start the assay two days after seeding.    -   8. Test compounds are dissolved in 4% DMSO. Samples are then        further diluted directly on plates with starve-DMEM. Typically,        this dilution will be 1:10 or greater. All wells are then        transferred to the cell plate at a further 1:10 dilution (10 μl        sample and media into 90 μl of starve media). The final DMSO        concentration should be 1% or lower. A standard serial dilution        may also be used.    -   9. Incubate under 5% CO₂ at 37° C. for 2 hours.    -   10. Prepare EGF ligand by diluting stock EGF (16.5 μM) in warm        DMEM to 150 nM.    -   11. Prepare fresh HNTG* sufficient for 100 μL per well; place on        ice.    -   12. After 2 hour incubation with test compound, add prepared EGF        ligand to cells, 50 μL per well, for a final concentration of 50        nM. Positive control wells receive the same amount of EGF.        Negative controls do not receive EGF. Incubate at 37° C. for 10        min.    -   13. Remove test compound, EGF, and DMEM. Wash cells once with        PBS.    -   14. Transfer HNTG* to cells, 100 μL per well. Place on ice for 5        minutes. Meanwhile, remove blocking buffer from ELISA plate and        wash.    -   15. Scrape cells from plate with a micropipettor and homogenize        cell material by repeatedly aspirating and dispensing the HNTG*        lysis buffer. Transfer lysate to a coated, blocked, washed ELISA        plate.    -   16. Incubate, with shaking, at room temperature for 1 hr.    -   17. Remove lysate, wash. Transfer freshly diluted anti-Ptyr        antibody (1:3000 in TBST) to ELISA plate, 100 μL per well.    -   18. Incubate, with shaking, at room temperature, for 30 min.    -   19. Remove anti-Ptyr antibody, wash. Transfer freshly diluted        BIOSOURCE antibody to ELISA plate(1:8000 in TBST, 100 μL per        well).    -   20. Incubate, with shaking, at room temperature for 30 min.    -   21. Remove BIOSOURCE antibody, wash. Transfer freshly prepared        ABTS/H₂O₂ solution to ELISA plate, 100 μL per well.    -   22. Incubate, with shaking, for 5–10 minutes. Remove any        bubbles.    -   23. Stop reaction by adding 100 μL of 0.2M HCl per well.    -   24. Read assay on Dynatech MR7000 ELISA reader with test filter        set at 410 nM and reference filter at 630 nM.        CDK2/Cyclin a Assay

This assay is used to measure the in vitro serine/threonine kinaseactivity of human cdk2/cyclin A in a Scintillation Proximity Assay(SPA).

Materials and Reagents.

-   -   1. Wallac 96-well polyethylene terephthalate (flexi) plates        (Wallac Catalog # 1450-401).    -   2. Amersham Redivue [γ³³P] ATP (Amersham catalog #AH 9968).    -   3. Amersham streptavidin coated polyvinyltoluene SPA beads        (Amersham catalog #RPNQ0007). The beads should be reconstituted        in PBS without magnesium or calcium, at 20 mg/ml.    -   4. Activated cdk2/cyclin A enzyme complex purified from Sf9        cells (SUGEN, Inc.).    -   5. Biotinylated peptide substrate (Debtide). Peptide        biotin-X-PKTPKKAKKL is dissolved in dH₂O at a concentration of 5        mg/ml.    -   6. 20% DMSO in dH₂O.    -   7. Kinase buffer: for 10 ml, mix 9.1 ml dH₂O, 0.5 ml TRIS(pH        7.4), 0.2 ml 1M MgCl₂, 0.2 ml 10% NP40 and 0.02 ml 1M DTT, added        fresh just prior to use.    -   8. 10 mM ATP in dH₂O.    -   9. 1M Tris, pH adjusted to 7.4 with HCl.    -   10. 1M MgCl₂.    -   11. 1M DTT.    -   12. PBS (Gibco Catalog # 14190-144).    -   13. 0.5M EDTA.    -   14. Stop solution: For 10 ml, mix 9.25 ml PBS, 0.05 ml 10 mM        ATP, 0.1 ml 0.5 M EDTA, 0.1 ml 10% Triton X-100 and 1.5 ml of 50        mg/ml SPA beads.        Procedure:    -   1. Prepare solutions of test compounds at 4× the desired final        concentration in 5% DMSO. Add 10 μL to each well. For positive        and negative controls, use 10 μL 20% DMSO alone in wells.    -   2. Dilute the peptide substrate (deb-tide) 1:250 with dH₂O to        give a final concentration of 0.02 mg/ml.    -   3. Mix 24 μL 0.1 mM ATP with 24 μCi γ³³P ATP and enough dH₂O to        make 600 μL.    -   4. Mix diluted peptide and ATP solutions 1:1 (600 μL+600 μL per        plate). Add 10 μL of this solution to each well.    -   5. Dilute 5 μL of cdk2/cyclin A solution into 2.1 ml 2× kinase        buffer (per plate). Add 20 μL enzyme per well. For negative        controls, add 20 μL 2× kinase buffer without enzyme.    -   6. Mix briefly on a plate shaker; incubate for 60 minutes.    -   7. Add 200 μL stop solution per well.    -   8. Let stand at least 10 min.    -   9. Spin plate at approx. 2300 rpm for 10–15 min.    -   10. Count plate on Trilux reader.        Met Transphosphorylation Assay

This assay is used to measure phosphotyrosine levels on a poly(glutamicacid:tyrosine, 4:1) substrate as a means for identifyingagonists/antagonists of met transphosphorylation of the substrate.

Materials and Reagents:

-   -   1. Corning 96-well ELISA plates, Corning Catalog # 25805-96.    -   2. Poly(glu-tyr), 4:1, Sigma, Cat. No; P 0275.    -   3. PBS, Gibco Catalog # 450-1300EB    -   4. 50 mM HEPES    -   5. Blocking Buffer: Dissolve 25 g Bovine Serum Albumin, Sigma        Cat. No A-7888, in 500 ml PBS, filter through a 4 μm filter.    -   6. Purified GST fusion protein containing the Met kinase domain,        SUGEN, Inc.    -   7. TBST Buffer.    -   8. 10% aqueous (MilliQue H₂O) DMSO.    -   9. 10 mM aqueous (dH₂O) Adenosine-5′-triphosphate, Sigma Cat.        No. A-5394.    -   10. 2× Kinase Dilution Buffer: for 100 ml, mix 10 mL 1M HEPES at        pH 7.5 with 0.4 mL 5% BSA/PBS, 0.2 mL 0.1 M sodium orthovanadate        and 1 mL 5M sodium chloride in 88.4 mL dH₂O.    -   11. 4×ATP Reaction Mixture: for 10 mL, mix 0.4 mL 1 M manganese        chloride and 0.02 mL 0.1 M ATP in 9.56 mL dH₂O.    -   12. 4× Negative Controls Mixture: for 10 mL, mix 0.4 mL 1 M        manganese chloride in 9.6 mL dH₂O.    -   13. NUNC 96-well V bottom polypropylene plates, Applied        Scientific Catalog # S-72092    -   14. 500 mM EDTA.    -   15. Antibody Dilution Buffer: for 100 mL, mix 10 mL 5% BSA/PBS,        0.5 mL 5% Carnation® Instant Milk in PBS and 0.1 mL 0.1 M sodium        orthovanadate in 88.4 mL TBST.    -   16. Rabbit polyclonal antophosphotyrosine antibody, SUGEN, Inc.    -   17. Goat anti-rabbit horseradish peroxidase conjugated antibody,        Biosource, Inc.    -   18. ABTS Solution: for 1 L, mix 19.21 g citric acid, 35.49 g        Na₂HPO₄ and 500 mg ABTS with sufficient dH₂O to make 1 L.    -   19. ABTS/H₂O₂: mix 15 mL ABST solution with 2 μL H₂O₂ five        minutes before use.    -   20. 0.2 M HCl        Procedure:    -   1. Coat ELISA plates with 2 μg Poly(Glu-Tyr) in 100 μL PBS, hold        overnight at 4° C.    -   2. Block plate with 150 μL of 5% BSA/PBS for 60 min.    -   3. Wash plate twice with PBS then once with 50 mM Hepes buffer        pH 7.4.    -   4. Add 50 μl of the diluted kinase to all wells. (Purified        kinase is diluted with Kinase Dilution Buffer. Final        concentration should be 10 ng/well.)    -   5. Add 25 μL of the test compound (in 4% DMSO) or DMSO alone (4%        in dH₂O) for controls to plate.    -   6. Incubate the kinase/compound mixture for 15 minutes.    -   7. Add 25 μL of 40 mM MnCl₂ to the negative control wells.    -   8. Add 25 μL ATP/MnCl₂ mixture to the all other wells (except        the negative controls). Incubate for 5 min.    -   9. Add 25 μL 500 mM EDTA to stop reaction.    -   10. Wash plate 3× with TBST.    -   11. Add 100 μL rabbit polyclonal anti-Ptyr diluted 1:10,000 in        Antibody Dilution Buffer to each well. Incubate, with shaking,        at room temperature for one hour.    -   12. Wash plate 3× with TBST.    -   13. Dilute Biosource HRP conjugated anti-rabbit antibody 1:6,000        in Antibody Dilution buffer. Add 100 μL per well and incubate at        room temperature, with shaking, for one hour.    -   14. Wash plate 1× with PBS.    -   15. Add 100 μl of ABTS/H₂O₂ solution to each well.    -   16. If necessary, stop the development reaction with the        addition of 100 μl of 0.2M HCl per well.    -   17. Read plate on Dynatech MR7000 ELISA reader with the test        filter at 410 nM and the reference filter at 630 nM.        IGF-1 Transphosphorylation Assay

This assay is used to measure the phosphotyrosine level in poly(glutamicacid:tyrosine, 4:1) for the identification of agonists/antagonists ofgst-IGF-1 transphosphorylation of a substrate.

Materials and Reagents:

-   -   1. Corning 96-well ELISA plates.    -   2. Poly(Glu-Tyr),4:1, Sigma Cat. No. P 0275.    -   3. PBS, Gibco Catalog # 450-1300EB.    -   4. 50 mM HEPES    -   5. TBB Blocking Buffer: for 1 L, mix 100 g BSA, 12.1 gTRIS (pH        7.5), 58.44 g sodium chloride and 10 mL 1% TWEEN-20.    -   6. Purified GST fusion protein containing the IGF-1 kinase        domain (SUGEN, Inc.)    -   7. TBST Buffer: for 1 L, mix 6.057 g Tris, 8.766 g sodium        chloride and 0.5 ml TWEEN-20 with enough dH₂O to make 1 liter.    -   8. 4% DMSO in Milli-Q H₂O.    -   9. 10 mM ATP in dH₂O.    -   10. 2× Kinase Dilution Buffer: for 100 mL, mix 10 mL 1 M HEPES        (pH 7.5), 0.4 mL 5% BSA in dH₂O, 0.2 mL 0.1 M sodium        orthovanadate and 1 mL 5 M sodium chloride with enough dH₂O to        make 100 mL.    -   11. 4×ATP Reaction Mixture: for 10 mL, mix 0.4 mL 1 M MnCl₂ and        0.008 mL 0.01 M ATP and 9.56 mL dH₂O.    -   12. 4× Negative Controls Mixture: mix 0.4 mL 1 M MnCl₂ in 9.60        mL dH₂O.    -   13. NUNC 96-well V bottom polypropylene plates.    -   14. 500 mM EDTA in dH₂O.    -   15. Antibody Dilution Buffer: for 100 mL, mix 10 mL 5% BSA in        PBS, 0.5 mL 5% Carnation Instant Non-fat Milk in PBS and 0.1 mL        0.1 M sodium orthovanadate in 88.4 mL TBST.    -   16. Rabbit Polyclonal antiphosphotyrosine antibody, SUGEN, Inc.    -   17. Goat anti-rabbit HRP conjugated antibody, Biosource.    -   18. ABTS Solution.    -   20. ABTS/H₂O₂: mix 15 mL ABTS with 2 μL H₂O₂ 5 minutes before        using.    -   21. 0.2 M HCl in dH₂O.        Procedure:    -   1. Coat ELISA plate with 2.0 μg/well Poly(Glu, Tyr), 4:1 (Sigma        P0275) in 100 μl PBS. Store plate overnight at 4° C.    -   2. Wash plate once with PBS.    -   3. Add 100 μl of TBB Blocking Buffer to each well. Incubate        plate for 1 hour with shaking at room temperature.    -   4. Wash plate once with PBS, then twice with 50 mM Hepes buffer        pH 7.5.    -   5. Add 25 μL of test compound in 4% DMSO (obtained by diluting a        stock solution of 10 mM test compound in 100% DMSO with dH₂O) to        plate.    -   6. Add 10.0 ng of gst-IGF-1 kinase in 50 μl Kinase Dilution        Buffer to all wells.    -   7. Start kinase reaction by adding 25 μl 4×ATP Reaction Mixture        to all test wells and positive control wells. Add 25 μl 4×        Negative Controls Mixture to all negative control wells.        Incubates for 10 minutes, with shaking, at room temperature.    -   8. Add 25 μl 0.5M EDTA (pH 8.0) to all wells.    -   9. Wash plate 4× with TBST Buffer.    -   10. Add rabbit polyclonal anti-phosphotyrosine antisera at a        dilution of 1:10,000 in 100 μl Antibody Dilution Buffer to all        wells. Incubate, with shaking, at room temperature for 1 hour.    -   11. Wash plate as in step 9.    -   12. Add 100 μL Biosource anti-rabbit HRP at a dilution of        1:10,000 in Antibody dilution buffer to all wells. Incubate,        with shaking, at room temperature for 1 hour.    -   13. Wash plate as in step 9, follow with one wash with PBS to        remove bubbles and excess Tween-20.    -   14. Develop by adding 100 μl/well ABTS/H₂O₂ to each well    -   15. After about 5 minutes, read on ELISA reader with test filter        at 410 nm and referenced filter at 630 nm.        BrdU Incorporation Assays

The following assays use cells engineered to express a selected receptorand then evaluate the effect of a compound of interest on the activityof ligand-induced DNA synthesis by determining BrdU incorporation intothe DNA.

The following materials, reagents and procedure are general to each ofthe following BrdU incorporation assays. Variances in specific assaysare noted.

General Materials and Reagents:

-   -   1. The appropriate ligand.    -   2. The appropriate engineered cells.    -   3. BrdU Labeling Reagent: 10 mM, in PBS, pH7.4(Roche Molecular        Biochemicals, Indianapolis, Ind.).    -   4. FixDenat: fixation solution (Roche Molecular Biochemicals,        Indianapolis, Ind.).    -   5. Anti-BrdU-POD: mouse monoclonal antibody conjugated with        peroxidase (Chemicon, Temecula, Calif.).    -   6. TMB Substrate Solution: tetramethylbenzidine (TMB, ready to        use, Roche Molecular Biochemicals, Indianapolis, Ind.).    -   7. PBS Washing Solution: 1×PBS, pH 7.4.    -   8. Albumin, Bovine (BSA), fraction V powder (Sigma Chemical Co.,        USA).        General Procedure:    -   1. Cells are seeded at 8000 cells/well in 10% CS, 2 mM Gln in        DMEM, in a 96 well plate. Cells are incubated overnight at        37° C. in 5% CO_(2.)    -   2. After 24 hours, the cells are washed with PBS, and then are        serum-starved in serum free medium (0% CS DMEM with 0.1% BSA)        for 24 hours.    -   3. On day 3, the appropriate ligand and the test compound are        added to the cells simultaneously. The negative control wells        receive serum free DMEM with 0.1% BSA only; the positive control        cells receive the ligand but no test compound. Test compounds        are prepared in serum free DMEM with ligand in a 96 well plate,        and serially diluted for 7 test concentrations.    -   4. After 18 hours of ligand activation, diluted BrdU labeling        reagent (1:100 in DMEM, 0.1% BSA) is added and the cells are        incubated with BrdU (final concentration is 10 μM) for 1.5        hours.    -   5. After incubation with labeling reagent, the medium is removed        by decanting and tapping the inverted plate on a paper towel.        FixDenat solution is added (50 μl/well) and the plates are        incubated at room temperature for 45 minutes on a plate shaker.    -   6. The FixDenat solution is removed by decanting and tapping the        inverted plate on a paper towel. Milk is added (5% dehydrated        milk in PBS, 200 μl/well) as a blocking solution and the plate        is incubated for 30 minutes at room temperature on a plate        shaker.    -   7. The blocking solution is removed by decanting and the wells        are washed once with PBS. Anti-BrdU-POD solution is added (1:200        dilution in PBS, 1% BSA, 50 μl/well) and the plate is incubated        for 90 minutes at room temperature on a plate shaker.    -   8. The antibody conjugate is removed by decanting and rinsing        the wells 5 times with PBS, and the plate is dried by inverting        and tapping on a paper towel.    -   9. TMB substrate solution is added (100 μl/well) and incubated        for 20 minutes at room temperature on a plate shaker until color        development is sufficient for photometric detection.    -   10. The absorbance of the samples are measured at 410 nm (in        “dual wavelength” mode with a filter reading at 490 nm, as a        reference wavelength) on a Dynatech ELISA plate reader.        EGF-Induced BrdU Incorporation Assay        Materials and Reagents:    -   1. Mouse EGF, 201 (Toyobo Co., Ltd., Japan).    -   2. 3T3/EGFRc7.        Remaining Materials and Reagents and Procedure, as above.        EGF-Induced Her-2-Driven BrdU Incorporation Assay        Materials and Reagents:    -   1. Mouse EGF, 201 (Toyobo Co., Ltd., Japan).    -   2. 3T3/EGFr/Her2/EGFr (EGFr with a Her-2 kinase domain).        Remaining Materials and Reagents and Procedure, as above.        EGF-Induced Her-4-Driven BrdU Incorporation Assay        Materials and Reagents:    -   1. Mouse EGF, 201 (Toyobo Co., Ltd., Japan).    -   2. 3T3/EGFr/Her4/EGFr (EGFr with a Her-4 kinase domain).        Remaining Materials and Reagents and Procedure, as above.        PDGF-Induced BrdU Incorporation Assay        Materials and Reagents:    -   1.Human PDGF B/B (Boehringer Mannheim, Germany).    -   2. 3T3/EGFRc7. Remaining Materials and Reagents and Procedure,        as above.        FGF-Induced BrdU Incorporation Assay        Materials and Reagents:    -   1. Human FGF2/bFGF (Gibco BRL, USA).    -   2. 3T3c7/EGFr Remaining Materials and Reagents and Procedure, as        above.        IGF1-Induced BrdU Incorporation Assay        Materials and Reagents:    -   1.Human, recombinant (G511, Promega Corp., USA)    -   2. 3T3/IGF1r. Remaining Materials and Reagents and Procedure, as        above.        Insulin-Induced BrdU Incorporation Assay        Materials and Reagents:    -   1. Insulin, crystalline, bovine, Zinc (13007, Gibco BRL, USA).    -   2. 3T3/H25. Remaining Materials and Reagents and Procedure, as        above.        HGF-Induced BrdU Incorporation Assay        Materials and Reagents:    -   1. Recombinant human HGF (Cat. No. 249-HG, R&D Systems, Inc.        USA).    -   2. BxPC-3 cells (ATCC CRL-1687). Remaining Materials and        Reagents, as above.        Procedure:    -   1. Cells are seeded at 9000 cells/well in RPMI 10% FBS in a 96        well plate. Cells are incubated overnight at 37° C. in 5% CO₂.    -   2. After 24 hours, the cells are washed with PBS, and then are        serum starved in 100 μl serum-free medium (RPMI with 0.1% BSA)        for 24 hours.    -   3. On day 3, 25 μl containing ligand (prepared at 1 μg/ml in        RPMI with 0.1% BSA; final HGF conc. is 200 ng/ml) and test        compounds are added to the cells. The negative control wells        receive 25 μl serum-free RPMI with 0.1% BSA only; the positive        control cells receive the ligand (HGF) but no test compound.        Test compounds are prepared at 5 times their final concentration        in serum-free RPMI with ligand in a 96 well plate, and serially        diluted to give 7 test concentrations. Typically, the highest        final concentration of test compound is 100 μM, and 1:3        dilutions are used (i.e. final test compound concentration range        is 0.137–100 μM).    -   4. After 18 hours of ligand activation, 12.5 μl of diluted BrdU        labeling reagent (1:100 in RPMI, 0.1% BSA) is added to each well        and the cells are incubated with BrdU (final concentration is 10        μM) for 1 hour.    -   5. Same as General Procedure.    -   6. Same as General Procedure.    -   7. The blocking solution is removed by decanting and the wells        are washed once with PBS. Anti-BrdU-POD solution (1:100 dilution        in PBS, 1% BSA) is added (100 μl/well) and the plate is        incubated for 90 minutes at room temperature on a plate shaker.    -   8. Same as General Procedure.    -   9. Same as General Procedure.    -   10. Same as General Procedure.        Exponential BrdU Incorporation Assay

This assay is used to measure the proliferation (DNA synthesis) ofexponentially growing A431 cells. The assay will screen for compoundsthat inhibit cell cycle progression.

Materials and Reagents:

Healthy growing A431 cells. The remainder of the Materials and Reagentsare the same as listed above in the general protocol section.

Procedure:

-   -   1. A431 cells are seeded at 8000 cells/well in 10% FBS, 2mM Gln        in DMEM, on a 96-well plate. Cells are incubated overnight at        37° C. in 5% CO₂.    -   2. On day 2, test compounds are serially diluted to 7 test        concentrations in the same growth medium on a 96-well plate and        then are added to the cells on a 96-well tissue culture plate.    -   3. After 20–24 hours of incubation, diluted BrdU labeling        reagent (1:100 in DMEM, 0.1% BSA) is added and the cells are        incubated with BrdU (final concentration is 10 μM) for 2 hours.

Steps 5–10 of the General Procedure are used to complete the assay.

ZenSrc Assay

This assay is used to screen for inhibitors of the tyrosine kinase Src.

Materials and Reagents:

-   -   1. Coating buffer: PBS containing sodium azide (0.2 mg/ml).    -   2. 1% w/v BSA in PBS.    -   3. Wash buffer: PBS containing 0.05% v/v Tween 20 (PBS-TWEEN)    -   4. 500 mM HEPES pH7.4.    -   5. ATP (40 μM)+MgCl₂ (80 mM) in distilled water.    -   6. MgCl₂ (80 mM) in distilled water (for no ATP blanks).    -   7. Test compounds, 10 mM in DMSO.    -   8. Assay Buffer: 100 mM HEPES, pH 7.4, containing 2 mM DTT, 0.2        mM sodium orthovanadate and 0.2 mgs/ml BSA.    -   9. Partially purified recombinant human Src (UBI (14–117)    -   10. Anti-phosphotyrosine (SUGEN rabbit polyclonal anti-PY).    -   11.HRP-linked goat anti-rabbit Ig (Biosource International        #6430)    -   12. HRP substrate ABTS or Pierce Peroxidase substrate.    -   13. Corning ELISA plates.        Procedure:    -   1. Coat plates with 100 μl of 20 μg/ml poly(Glu-Tyr) (Sigma Cat.        No.P0275) containing 0.01% sodium azide. Hold overnight at 4° C.    -   2. Block with 1% BSA at 100 μl/well for one hour at room        temperature.    -   3. Plate test compounds (10 mM in DMSO) at 2 ul/well on a Costar        plate ready for dilution with dH₂O and plating to reaction        plates.    -   4. Dilute Src kinase 1:10,000 in Reaction Buffer, for 5 plates        prepare 25 ml as follows: 2.5 mls 1M HEPES pH7.4 (stored sterile        at 4° C.), 21.85 ml distilled water, 0.1 ml 5% BSA, 0.5 ml 10 mM        sodium orthovanadate (stored sterile at 4° C.), 50 μl 1.0M DTT        (stored frozen at −20° C.), and 2.5 μl Src Kinase (stored frozen        at −80° C.).    -   5. Add 48 μl of distilled water to the 2 μl of each compound in        the dilution plate then add 25 μl/well of this to the reaction        plate.    -   6. Add 50 μl of HRP to each reaction buffer well and then 25 μl        ATP-MgCl₂/well (MgCl₂ only to no ATP blanks). Incubate at room        temperature for 15 minutes on plate shaker. Stop reaction by        adding 25 μl of 0.5M EDTA to each well.    -   7. Wash 4× with PBS-TWEEN.    -   8. Add 100 μl anti-phosphotyrosine (1:10,000 of anti-pTyr serum        or 1:3,000 of 10% glycerol diluted PA-affinity purified        antibody) in PBS-TWEEN containing 0.5% BSA, 0.025% Non-fat milk        powder and 100 μM sodium orthovanadate. Incubate with continuous        shaking at room temperature for one hour.    -   9. Wash plates 4× with PBS-TWEEN.    -   10. Add 100 μl HRP-linked Ig (1:5,000) in PBS-TWEEN containing        0.5% BSA, 0.025% Non-fat milk powder, 100 μM sodium        orthovanadate. Incubate with shaking at room temperature for one        hour.    -   11. Wash plates 4× with PBS-TWEEN and then once with PBS.    -   12. Develop plate using ABTS or other peroxidase substrate.        Cell Cycle Analysis:

A431 cells in standard growth medium are exposed to a desiredconcentration of a test compound for 20–24 hours at 37° C. The cells arethen collected, suspended in PBS, fixed with 70% ice-cold methanol andstained with propidium iodide. The DNA content is then measured using aFACScan flow cytometer. Cell cycle phase distribution can then beestimated using CellFIT software (Becton-Dickinson).

HUV-EC-C Assay

This assay is used to measure a compound's activity against PDGF-R,FGF-R, VEGF, aFGF or Flk-1/KDR, all of which are naturally expressed byHUV-EC cells.

Day 0

1. Wash and trypsinize HUV-EC-C cells (human umbilical vein endothelialcells, (American Type Culture Collection, catalogue no. 1730 CRL). Washwith Dulbecco's phosphate-buffered saline (D-PBS, obtained from GibcoBRL, catalogue no. 14190-029) 2 times at about 1 ml/10 cm² of tissueculture flask. Trypsinize with 0.05% trypsin-EDTA in non-enzymatic celldissociation solution (Sigma Chemical Company, catalogue no. C-1544).The 0.05% trypsin is made by diluting 0.25% trypsin/1 mM EDTA (Gibco,catalogue no.25200-049) in the cell dissociation solution. Trypsinizewith about 1 ml/25–30 cm² of tissue culture flask for about 5 minutes at37° C. After cells have detached from the flask, add an equal volume ofassay medium and transfer to a 50 ml sterile centrifuge tube (FisherScientific, catalogue no. 05-539-6).

2. Wash the cells with about 35 ml assay medium in the 50 ml sterilecentrifuge tube by adding the assay medium, centrifuge for 10 minutes atapproximately 200×g, aspirate the supernatant, and resuspend with 35 mlD-PBS. Repeat the wash two more times with D-PBS, resuspend the cells inabout 1 ml assay medium/15 cm² of tissue culture flask. Assay mediumconsists of F12K medium (Gibco BRL, catalogue no. 21127-014) and 0.5%heat-inactivated fetal bovine serum. Count the cells with a CoulterCounter® (Coulter Electronics, Inc.) and add assay medium to the cellsto obtain a concentration of 0.8–1.0×10⁵ cells/ml.

3. Add cells to 96-well flat-bottom plates at 100 μl/well or 0.8–1.0×10⁴cells/well, incubate ˜24 h at 37° C., 5% CO₂.

Day 1

1. Make up two-fold test compound titrations in separate 96-well plates,generally 50 μM on down to 0 μM. Use the same assay medium as mentionedin day 0, step 2 above. Titrations are made by adding 90 μl/well of testcompound at 200 μM (4× the final well concentration) to the top well ofa particular plate column. Since the stock test compound is usually 20mM in DMSO, the 200 μM drug concentration contains 2% DMSO.

A diluent made up to 2% DMSO in assay medium (F12K+0.5% fetal bovineserum) is used as diluent for the test compound titrations in order todilute the test compound but keep the DMSO concentration constant. Addthis diluent to the remaining wells in the column at 60 μl/well. Take 60μl from the 120 μl of 200 μM test compound dilution in the top well ofthe column and mix with the 60 μl in the second well of the column. Take60 μl from this well and mix with the 60 μl in the third well of thecolumn, and so on until two-fold titrations are completed. When thenext-to-the-last well is mixed, take 60 μl of the 120 μl in this welland discard it. Leave the last well with 60 μl of DMSO/media diluent asa non-test compound-containing control. Make 9 columns of titrated testcompound, enough for triplicate wells each for: (1) VEGF (obtained fromPepro Tech Inc., catalogue no. 100–200, (2) endothelial cell growthfactor (ECGF) (also known as acidic fibroblast growth factor, or aFGF)(obtained from Boehringer Mannheim Biochemica, catalogue no. 1439 600),or, (3) human PDGF B/B (1276-956, Boehringer Mannheim, Germany) andassay media control. ECGF comes as a preparation with sodium heparin.

2. Transfer 50 μl/well of the test compound dilutions to the 96-wellassay plates containing the 0.8–1.0×10⁴ cells/100 μl/well of theHUV-EC-C cells from day 0 and incubate ˜2 h at 37° C., 5% CO₂.

3. In triplicate, add 50 μl/well of 80 μg/ml VEGF, 20 ng/ml ECGF, ormedia control to each test compound condition. As with the testcompounds, the growth factor concentrations are 4× the desired finalconcentration. Use the assay media from day 0 step 2 to make theconcentrations of growth factors. Incubate approximately 24 hours at 37°C., 5% CO₂. Each well will have 50 μl test compound dilution, 50 μlgrowth factor or media, and 100 μl cells, which calculates to 200μl/well total. Thus the 4× concentrations of test compound and growthfactors become 1× once everything has been added to the wells.

Day 2

1. Add 3H-thymidine (Amersham, catalogue no. TRK-686) at 1 μCi/well (10μl/well of 100 μCi/ml solution made up in RPMI media+10%heat-inactivated fetal bovine serum) and incubate ˜24 h at 37° C., 5%CO₂. RPMI is obtained from Gibco BRL, catalogue no. 11875-051.

Day 3

1. Freeze plates overnight at −20° C.

Day 4

Thaw plates and harvest with a 96-well plate harvester (Tomtec Harvester96®) onto filter mats (Wallac, catalogue no. 1205-401), read counts on aWallac Betaplate™ liquid scintillation counter.

Vascular Permeability Assay

Increased vascular permeability in tumor-dependent angiogenesis is dueto a loosening of gap junctions in response to vascular endothelialgrowth factor (VEGF). The Miles assay for vascular permeability (Milesand Miles, J. Physiol. 118: 228–257 (1952)) has been adapted to athymicmice in order to evaluate the ability of the compounds of the presentinvention to inhibit VEGF-induced vascular permeability in vivo.

General Procedure:

Test compound or vehicle is administered prior to (typically it is 4hours prior) to VEGF injection. 100 μl of 0.5% Evan's blue dye in PBS isinjected intravenously via lateral tail vein injections using a 27 gaugeneedle. Sixty minutes later, animals are anesthetized using the inhalantIsofluorane. Following anesthesia, VEGF (100 ng of VEGF in 20 μl of PBS)is injected intradermally in two spots and PBS (20 μl) is injected intwo spots in a grid pattern in the back of each animal. At a designatedtimepoint of up to 1 hour after VEGF injection, the animals areeuthanized by CO₂ and the skin patches are dissected and photographed.Based on a published report (Alicieri et al., Mol. Cell 4: 915–914(1999)) quantitative evaluation of the VEGF-dependent dye leakage intomouse skin can be achieved following elution of the dye from skinpatches.

In Vivo Animal Models

Xenograft Animal Models

The ability of human tumors to grow as xenografts in athymic mice (e.g.,Balb/c, nu/nu) provides a useful in vivo model for studying thebiological response to therapies for human tumors. Since the firstsuccessful xenotransplantation of human tumors into athymic mice,(Rygaard and Povlsen, 1969, Acta Pathol. Microbial. Scand. 77:758–760),many different human tumor cell lines (e.g., mammary, lung,genitourinary, gastro-intestinal, head and neck, glioblastoma, bone, andmalignant melanomas) have been transplanted and successfully grown innude mice. The following assays may be used to determine the level ofactivity, specificity and effect of the different compounds of thepresent invention. Three general types of assays are useful forevaluating compounds: cellular/catalytic, cellular/biological and invivo. The object of the cellular/catalytic assays is to determine theeffect of a compound on the ability of a TK to phosphorylate tyrosineson a known substrate in a cell. The object of the cellular/biologicalassays is to determine the effect of a compound on the biologicalresponse stimulated by a TK in a cell. The object of the in vivo assaysis to determine the effect of a compound in an animal model of aparticular disorder such as cancer.

Suitable cell lines for subcutaneous xenograft experiments include C6cells (glioma, ATCC # CCL 107), A375 cells (melanoma, ATCC # CRL 1619),A431 cells (epidermoid carcinoma, ATCC # CRL 1555), Calu 6 cells (lung,ATCC # HTB 56), PC3 cells (prostate, ATCC # CRL 1435), SKOV3TP5 cellsand NIH 3T3 fibroblasts genetically engineered to overexpress EGFR,PDGFR, IGF-1R or any other test kinase. The following protocol can beused to perform xenograft experiments:

Female athymic mice (BALB/c, nu/nu) are obtained from SimonsenLaboratories (Gilroy, Calif.). All animals are maintained underclean-room conditions in Micro-isolator cages with Alpha-dri bedding.They receive sterile rodent chow and water ad libitum.

Cell lines are grown in appropriate medium (for example, MEM, DMEM,Ham's F10, or Ham's F12 plus 5%-10% fetal bovine serum (FBS) and 2 mMglutamine (GLN)). All cell culture media, glutamine, and fetal bovineserum are purchased from Gibco Life Technologies (Grand Island, N.Y.)unless otherwise specified. All cells are grown in a humid atmosphere of90–95% air and 5–10% CO₂ at 37° C. All cell lines are routinelysubcultured twice a week and are negative for mycoplasma as determinedby the Mycotect method (Gibco).

Cells are harvested at or near confluency with 0.05% Trypsin-EDTA andpelleted at 450×g for 10 min. Pellets are resuspended in sterile PBS ormedia (without FBS) to a particular concentration and the cells areimplanted into the hindflank of the mice (8–10 mice per group, 2–10×10⁶cells/animal). Tumor growth is measured over 3 to 6 weeks using veniercalipers. Tumor volumes are calculated as a product oflength×width×height unless otherwise indicated. P values are calculatedusing the Students t-test. Test compounds in 50–100 μL excipient (DMSO,or VPD:D5W) can be delivered by IP injection at different concentrationsgenerally starting at day one after implantation.

Tumor Invasion Model

The following tumor invasion model has been developed and may be usedfor the evaluation of therapeutic value and efficacy of the compoundsidentified to selectively inhibit KDR/FLK-1 receptor.

Procedure

8 week old nude mice (female) (Simonsen Inc.) are used as experimentalanimals. Implantation of tumor cells can be performed in a laminar flowhood. For anesthesia, Xylazine/Ketamine Cocktail (100 mg/kg ketamine and5 mg/kg Xylazine) are administered intraperitoneally. A midline incisionis done to expose the abdominal cavity (approximately 1.5 cm in length)to inject 10⁷ tumor cells in a volume of 100 μl medium. The cells areinjected either into the duodenal lobe of the pancreas or under theserosa of the colon. The peritoneum and muscles are closed with a 6–0silk continuous suture and the skin is closed by using wound clips.Animals are observed daily.

Analysis

After 2–6 weeks, depending on gross observations of the animals, themice are sacrificed, and the local tumor metastases to various organs(lung, liver, brain, stomach, spleen, heart, muscle) are excised andanalyzed (measurement of tumor size, grade of invasion, immunochemistry,in situ hybridization determination, etc.).

Additional Assays

Additional assays which may be used to evaluate the compounds of thisinvention include, without limitation, a bio-flk-1 assay, an EGFreceptor-HER2 chimeric receptor assay in whole cells, a bio-src assay, abio-lck assay and an assay measuring the phosphorylation function ofraf. The protocols for each of these assays may be found in U.S.application Ser. No. 09/099,842, which is incorporated by reference,including any drawings, herein. Additionally, U.S. Pat. No. 5,792,783,filed Jun. 5, 1996 and U.S. application Ser. No. 09/322,297, filed May28, 1999 are incorporated by reference as if fully set forth herein.

Plasma Stability Test:

The prodrug(3Z)-3-[(3,5-dimethyl-1H-pyrrol-2-yl)-methylidene]-1-(1-pyrrolidinylmethyl)-1,3-dihydro-2H-indol-2-onewas administered IV at 2 mg/mL to dogs. Levels of both prodrug and drug(3(Z)-3-[(3,5-dimethyl-1H-pyrrol-2-yl)-methylidene]-1,3-dihydro-2H-indol-2-one)were followed by HPLC analysis of blood plasma for 4 hours followingdosing. This study showed that the half-life for conversion of theprodrug to drug was 7.3 min. From a plot of drug concentration vs. time,the area under the curve indicated that 80% of the prodrug was convertedinto drug.

Formulation Examples

The formulations being evaluated are listed in Tables 1 and 2 and aredescribed below:

[A] Solid formulations to be reconstituted to a stable infusate (Table1):

(1) Lyophilized Formulation:

-   -   (a) Captisol Based: This formulation uses Captisol and an acidic        agent to form an in situ salt at a pH of 1.5–2.0 to compound and        lyophilize solutions of drug at concentrations of 20.0–25.0        mg/mL. The lyophilized cake is reconstituted with an IV fluid to        provide a stable infusate at 2 mg/mL or higher at pH 3.    -   (b) Non-Captisol based: This formulation uses small amounts of a        surfactant such as Polysorbate-80 or Cremophor EL and an acidic        agent to form an in-situ salt at a pH of 1.5–2.0 to compound and        lyophilize solutions of drug at concentrations of 20.0–25.0        mg/mL. The lyophilized cake is reconstituted with        cosolvent-surfactant based aqueous diluent such as        PEG-300-Polysorbate 80 or PEG-300-Cremophor EL to provide a        stable infusate at 2 mg/mL or higher at pH 3.        (2) Sterile API fill:    -   The drug is filled as a sterile powder fill in a container and        will be reconstituted with a specific co-solvent—surfactant        based aqueous diluent to provide a stable infusate at 2 mg/mL or        higher of drug at pH 3.        [B] Solution concentrate to be diluted to a stable infusate        (Table 2):    -   The drug is solubilized in a non-aqueous mixture of co-solvents        and surfactants at a high concentration such that it can be        diluted with aqueous diluents to a stable infusate. The        concentration of the drug in the infusate is at a concentration        of 2 mg/mL or higher, at pH 3. The total levels of the        co-solvent is less than 15% and the levels of surfactant is than        0.5%.

TABLE 1 Formulation (1) Solid formulations Attributes Lyophilized -Captisol based Lyophilized Non-Captisol based Sterile API Fill Dose/50CC 200–300 200–300 300–400 vial (mgs) Sterile API fill NA NA 300–400 mgin vial Composition - drug (mg) 200–300 drug (mg) 200–300 NA LyophilizedAcid (M) 1.4 Acid (M) 1.4 Cake Antioxidant (mg)  0–10 Antioxidant (mg) 0–10 Captisol (mg) 2000–3000 Filler (mg) 200–300 Polysorbate-80(mg) 0–50 Composition - 0.9% NaCl, D5W, PEG-300 (% w/v)  5–20 PEG-300 (%w/v)  5–20 Reconstitution Polysorbate-80 (% w/v)   0–1.0 Polysorbate-80(% w/v)   0–1.0 Fluid Citrate Buffer pH 3.0 30–40 Citrate Buffer pH 3.030–40 0.1M (% w/v) 0.1M (% w/v) Water (qs to volume) Water (qs tovolume) Composition - drug (mg/mL) 2–3 drug (mg/mL) 2–3 drug (mg/mL) 2–3Reconstituted Acid (Molar) 1.4 Acid (Molar) 1.4 Acid (Molar) 1.4Infusate Antioxidant (mg/mL)   0–1.0 Antioxidant (mgmL)   0–1.0Antioxidant (mg/mL)   0–1.0 (Administered Captisol (mg/mL) 20–30 Filler(mg/mL) 2–3 Filler (mg/mL) 2–3 to Patient) IV fluid (qs to volume) 3.0PEG-300 (mg/mL)  50–200 PEG-300 (mg/mL)  50–200 pH Polysorbate-80(mg/mL)  0–10 Polysorbate-80 (mg/mL)  0–10 Citrate Buffer 0.3 CitrateBuffer 0.3 0.1M, pH 3.0 (mL) 0.1M, pH 3.0 (mL) Water (qs to volume)Water (qs to volume) pH 3.0 pH 3.0 Composition - drug (mg/mL) 20–30 drug(mg/mL) 20–30 NA Prelyophilate Acid (Molar) 1.4 Acid (Molar) 1.4Antioxidant (mg/mL)  0–10 Antioxidant (mg/mL)  0–10 Captisol (mg/mL)200–300 Filler (mg/mL) 20–30 Water for Injection qs to 1.0 mLPolysorbate-80(mg)  0–50 pH 1.5–2.0 Water for Injection qs to 1.0 mL pH1.5–2.0 Acids used: Methane sulfonic acid, Tartaric acid, Citric acid,succinic acid is used in a 1:1.4 molar ratio for in situ salt formationCosolvents- PEG-300, PEG-400 Surfactants: Polysorbate-80, Cremophor ELCaptisol ®: Sulfobutylether Cyclodextrin Drug: Compound of Example 2

TABLE 2 Composition of Solution Formulation Composition (% w/v) orIngredients mg/mL Drug 1.2–2.5 (12–25 mg/mL) Cosolvent - (PEG-300, PEG- 70–90 400) Surfactant   0–10 Dimethylacetamide   0–2 Salt forming agent(Methane Equivalent to 1.4 M ratio of sulfonic acid, Tartaric acid, theAPI Citric Acid, Succinic Acid) Alcohol Qs to VolumeFormulation to be diluted 10 fold with pharmaceutically acceptable IVfluids.

The present invention is not to be limited in scope by the exemplifiedembodiments which are intended as illustrations of single aspects of theinvention, and any clones, DNA or amino acid sequences which arefunctionally equivalent are within the scope of the invention. Indeed,various modifications of the invention in addition to those describedherein will become apparent to those skilled in the art from theforegoing description and accompanying drawings. Such modifications areintended to fall within the scope of the appended claims.

All references cited herein are hereby incorporated by reference intheir entirety.

1. A compound of the formula (I):

wherein: R² is hydrogen; R³, R⁴, R⁵ and R⁶ are independently selectedfrom the group consisting of hydrogen, alkyl, trihaloalkyl, cycloalkyl,alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy,aryloxy, mercapto, alkylthio, arylthio, sulfinyl, sulfonyl,S-sulfonamido, N-sulfonamido, trihalomethane-sulfonamido, carbonyl,C-carboxy, O-carboxy, C-amido, N-amido, cyano, nitro, halo, O-carbamyl,N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, amino and —NR¹¹R¹² where R¹¹and R¹² are independently selected from the group consisting ofhydrogen, alkyl, cycloalkyl, aryl, carbonyl, acetyl, sulfonyl, andtrifluoromethanesulfonyl, or R¹¹ and R¹² together with the nitrogen atomto which they are attached combine to form a five- or six-memberedheteroalicyclic ring provided that at least two of R³, R⁴, R⁵ and R⁶ arehydrogen; or R³ and R⁴, R⁴ and R⁵, or R⁵ and R⁶ combine to form asix-membered aryl ring, a methylenedioxy or an ethylenedioxy group; R⁷isselected from the group consisting of hydrogen, alkyl, cycloalkyl,alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy,aryloxy, carbonyl, acetyl, C-amido, C-thioamido, amidino, C-carboxy,O-carboxy, sulfonyl and trihalomethane-sulfonyl; R⁸ and R¹⁰ areunsubstituted lower alkyl; R⁹ is 2-(dimethylaminoethyl)aminocarbonyl,2-(diethylaminoethyl)aminocarbonyl,2-(pyrrolidin-1-ylethyl)aminocarbonyl, or2-(morpholin-4-ylethyl)aminocarbonyl; R^(1′) is hydrogen or alkyl; andR^(2′) is hydrogen, alkyl, aralkyl, acyl or —P(O)(OR)(OR′) where R andR′ are independently selected from the group consisting of hydrogen,alkyl, aralkyl or aryl; or a pharmaceutically acceptable salt thereof.2. The compound of claim 1, wherein R⁸ and R¹⁰ are each independentlymethyl.
 3. The compound of claim 1, wherein R^(2′) is hydrogen, acyl or—P(O)(OR)(OR′) and R⁷ is hydrogen; R³ is hydrogen or lower unsubstitutedalkyl; R⁴ is selected from the group consisting of hydrogen, halogen,aryl and S-sulfonamido; R⁵ is selected from the group consisting ofhydrogen, lower alkyl, lower alkoxy, aryl, and heteroaryl; and R⁶ ishydrogen.
 4. The compound of claim 3, wherein R³ is hydrogen or methyl.5. The compound of claim 3, wherein R⁴ is hydrogen, chloro, fluoro,bromo or phenyl.
 6. The compound of claim 5, wherein R⁴ is hydrogen orfluoro.
 7. The compound of claim 3, wherein R⁵ is hydrogen, methyl,ethyl, methoxy, phenyl or pyridyl.
 8. The compound of claim 7, whereinR⁵ is hydrogen.
 9. The compound of claim 1, wherein R^(2′) is hydrogen.10. The compound of claim 1, wherein R^(2′) is —P(O)(OR)(OR′).
 11. Thecompound of claim 1, wherein R^(2′) is acyl.
 12. A compound of theformula (II):

wherein: R² is hydrogen; R³, R⁴, R⁵ and R⁶ are independently selectedfrom the group consisting of hydrogen, alkyl, trihaloalkyl, cycloalkyl,alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy,aryloxy, mercapto, alkylthio, arylthio, sulfinyl, sulfonyl,S-sulfonamido, N-sulfonamido, trihalomethane-sulfonamido, carbonyl,C-carboxy, O-carboxy, C-amido, N-amido, cyano, nitro, halo, O-carbamyl,N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, amino and —NR¹¹R¹² where R¹¹and R¹² are independently selected from the group consisting ofhydrogen, alkyl, cycloalkyl, aryl, carbonyl, acetyl, sulfonyl, andtrifluoromethanesulfonyl, or R¹¹ and R¹² together with the nitrogen atomto which they are attached combine to form a five- or six-memberedheteroalicyclic ring provided that at least two of R³, R⁴, R⁵ and R⁶ arehydrogen; or R³ and R⁴, R⁴ and R⁵, or R⁵ and R⁶ combine to form asix-membered aryl ring, a methylenedioxy or an ethylenedioxy group; R⁷is selected from the group consisting of hydrogen, alkyl, cycloalkyl,alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy,aryloxy, carbonyl, acetyl, C-amido, C-thioamido, amidino, C-carboxy,O-carboxy, sulfonyl and trihalomethane-sulfonyl; R⁸ and R¹⁰ areindependently unsubstituted lower alkyl; R⁹ is —C(═O)NHR¹³ wherein R¹³is lower alkyl substituted with amino or heteroalicyclic and optionallysubstituted with hydroxy; R^(1′) is hydrogen or alkyl; and R^(3′) andR^(4′) are independently alkyl or together with the nitrogen atom towhich they are attached combine to form a heteroalicyclic ring or aheteroaryl ring; or a pharmaceutically acceptable salt thereof.
 13. Thecompound of claim 12, wherein R⁹ is (2-diethylaminoethyl)-aminocarbonyl,(2-ethylaminoethyl)aminocarbonyl,2-(pyrrolidin-1-ylethyl)-aminocarbonyl,3-(morpholin-4-yl)propyl-aminocarbonyl, or3-(morpholin-4-yl)-2-hydroxypropylaminocarbonyl.
 14. The compound ofclaim 13, wherein R⁹ is (2-diethylaminoethyl)aminocarbonyl, or(2-ethylaminoethyl)-aminocarbonyl.
 15. A compound of the formula II:

wherein: R³ is hydrogen or lower unsubstituted alkyl; R⁴ is selectedfrom the group consisting of hydrogen, halogen, aryl and S-sulfonamido;R⁵is selected from the group consisting of hydrogen, lower alkyl, loweralkoxy, aryl, and heteroaryl; R⁶ is hydrogen; R⁷ is hydrogen; R^(1′) ishydrogen or methyl R⁸ and R¹⁰ are independently unsubstituted loweralkyl; R⁹ is —C(═O)NHR¹³ wherein R¹³ is lower alkyl substituted withamino or heteroalicyclic and optionally substituted with hydroxy; andR^(3′) and R^(4′) are independently lower alkyl optionally substitutedwith hydroxy, or R^(3′) and R^(4′) together with the nitrogen atom towhich they are attached form a pyrrolidin-1-yl,2-(S)-hydroxymethylpyrrolidin-1-yl, 2-(S)-carboxy-pyrrolidin-1-yl,piperazin-1-yl, or 4-methylpiperazin-1-yl group; or R^(3′) and R^(4′)together with the nitrogen atom to which they are attached form aheteroaryl ring; or a pharmaceutically acceptable salt thereof.
 16. Thecompound of claim 15, wherein R^(3′) and R^(4′) are lower alkyloptionally substituted with hydroxyl.
 17. The compound of claim 15,wherein R^(3′) and R^(4′) together with the nitrogen atom to which theyare attached form a pyrrolidin-1-yl, 2-(S)-hydroxymethylpyrrolidin-1-yl,2-(S)-carboxy-pyrrolidin-1-yl, piperazin-1-yl, or a4-methylpiperazin-1-yl group.
 18. The compound of claim 17, whereinR^(3′) and R^(4′) together with the nitrogen atom to which they areattached form a pyrrolidin-1-yl group.
 19. The compound of claim 15,wherein R^(3′) and R^(4′) together with the nitrogen atom to which theyare attached form a pyrro-1-yl, pyridin-1-yl, oxazol-3-yl,isoxazol-2-yl, pyrazin-1-yl, pyradizin-1-yl, quinolin-1-yl, or aimidazol-1-yl heteroaryl ring.
 20. The compound of claim 19, whereinR^(3′) and R^(4′) together with the nitrogen atom to which they areattached form a pyridin-1-yl ring.
 21. The compound of claim 15, whereinR³ is hydrogen or methyl.
 22. The compound of claim 15, wherein R⁴ ishydrogen, chloro, fluoro, bromo or phenyl.
 23. The compound of claim 22,wherein R⁴ is hydrogen or fluoro.
 24. The compound of claim 15, whereinR⁵ is hydrogen, methyl, ethyl, methoxy, phenyl or pyridyl.
 25. Thecompound of claim 24, wherein R⁵ is hydrogen.
 26. The compound of claim15, wherein: R^(1′), R³, R⁵, R⁶, and R⁷ are hydrogen; R⁴ is halo; R⁸ andR¹⁰ are unsubstituted lower alkyl; R⁹ is —C(═O)NHR¹³ wherein R¹³ islower alkyl substituted with amino or heteroalicyclic and optionallysubstituted with hydroxyl; and R^(3′) and R^(4′) together with thenitrogen atom to which they are attached form a pyrrolidin-1-yl,2-(S)-hydroxymethylpyrrolidin-1-yl, 2-(S)-carboxypyrrolidin-1-yl,piperazin-1-yl, or a 4-methylpiperazin-1-yl group.
 27. The compound ofclaim 26, wherein R⁹ is (2-diethylaminoethyl)-aminocarbonyl,(2-ethylaminoethyl)-aminocarbonyl,2-(pyrrolidin-1-ylethyl)aminocarbonyl,3-(morpholin-4-yl)propyl-aminocarbonyl,3-(morpholin-4-yl)-2-hydroxypropylaminocarbonyl, particularly(2-diethylaminoethyl)aminocarbonyl, or(2-ethylaminoethyl)-aminocarbonyl.
 28. The compound of claim 27, whereinR⁹ is (2-diethylaminoethyl)aminocarbonyl, or(2-ethylaminoethyl)-aminocarbonyl.
 29. The compound of claim 27, whereinR^(3′) and R^(4′) together with the nitrogen atom to which they areattached form a pyrrolidin-1-yl group.
 30. The compound of claim 15,which is(3Z)-3-{[3,5-dimethyl-4-(2-diethylaminoethylaminocarbonyl)-1H-pyrrol-2-yl]-methylidene}-1-(1-pyrrolidinylmethyl)-1,3-dihydro-2H-indol-2-one;(3Z)-3-{[3,5-dimethyl-4-(2-ethylaminoethylaminocarbonyl)-1H-pyrrol-2-yl]-methylidene}-1-(1-pyrrolidinylmethyl)-1,3-dihydro-2H-indol-2-one;or(3Z)-3-{[3,5-dimethyl-4-(3-morpholin-4-yl-2-hydroxypropylaminocarbonyl)-1H-pyrrol-2-yl]-methylidene}-1-(1-pyrrolidinylmethyl)-1,3-dihydro-2H-indol-2-one.31. A compound of the formula III:

wherein: R² is hydrogen; R³, R⁴, R⁵ and R⁶ are independently selectedfrom the group consisting of hydrogen, alkyl, trihaloalkyl, cycloalkyl,alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy,aryloxy, mercapto, alkylthio, arylthio, sulfinyl, sulfonyl,S-sulfonamido, N-sulfonamido, trihalomethane-sulfonamido, carbonyl,C-carboxy, O-carboxy, C-amido, N-amido, cyano, nitro, halo, O-carbamyl,N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, amino and —NR¹¹R¹² where R¹¹and R¹² are independently selected from the group consisting ofhydrogen, alkyl, cycloalkyl, aryl, carbonyl, acetyl, sulfonyl, andtrifluoromethanesulfonyl, or R¹¹ and R¹² together with the nitrogen atomto which they are attached combine to form a five- or six-memberedheteroalicyclic ring provided that at least two of R³, R⁴, R⁵ and R⁶ arehydrogen; or R³ and R⁴, R⁴ and R⁵, or R⁵ and R⁶ combine to form asix-membered aryl ring, a methylenedioxy or an ethylenedioxy group; R⁷is selected from the group consisting of hydrogen, alkyl, cycloalkyl,alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy,aryloxy, carbonyl, acetyl, C-amido, C-thioamido, amidino, C-carboxy,O-carboxy, sulfonyl and trihalomethane-sulfonyl; R⁸ and R¹⁰ areunsubstituted lower alkyl; R⁹ is C-amido; and R^(5′) is alkyl; or apharmaceutically acceptable salt thereof.
 32. The compound of claim 31,wherein R⁹ is 2-(dimethylaminoethyl) aminocarbonyl,2-(diethylaminoethyl)aminocarbonyl,2-(pyrrolidin-1-ylethyl)aminocarbonyl, or2-(morpholin-4-ylethyl)aminocarbonyl.
 33. The compound of claim 31,wherein R³ is hydrogen or lower unsubstituted alkyl; R⁴ is selected fromthe group consisting of hydrogen, halogen, aryl and S-sulfonamido; R⁵ isselected from the group consisting of hydrogen, lower alkyl, loweralkoxy, aryl, and heteroaryl; and R⁶ and R⁷ are hydrogen.
 34. Thecompound of claim 31, wherein R³is hydrogen or methyl.
 35. The compoundof claim 31, wherein R⁴ is hydrogen, chloro, fluoro, bromo or phenyl.36. The compound of claim 35, wherein R⁴ is hydrogen or fluoro.
 37. Thecompound of claim 31, wherein R⁵ is hydrogen, methyl, ethyl, methoxy,phenyl or pyridyl.
 38. The compound of claim 37, wherein R⁵ is hydrogen.39. A compound of the formula IV:

wherein: R² is hydrogen; R³, R⁴, R⁵ and R⁶ are independently selectedfrom the group consisting of hydrogen, alkyl, trihaloalkyl, cycloalkyl,alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy,aryloxy, mercapto, alkylthio, arylthio, sulfinyl, sulfonyl,S-sulfonamido, N-sulfonamido, trihalomethane-sulfonamido, carbonyl,C-carboxy, O-carboxy, C-amido, N-amido, cyano, nitro, halo, O-carbamyl,N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, amino and —NR¹¹R¹² where R¹¹and R¹² are independently selected from the group consisting ofhydrogen, alkyl, cycloalkyl, aryl, carbonyl, acetyl, sulfonyl, andtrifluoromethanesulfonyl or R¹¹ and R¹² together with the nitrogen atomto which they are attached combine to form a five- or six-memberedheteroalicyclic ring provided that at least two of R³, R⁴, R⁵ and R⁶ arehydrogen; or R³ and R⁴, R⁴ and R⁵, or R⁵ and R⁶ combine to form asix-membered aryl ring, a methylenedioxy or an ethylenedioxy group; R⁷is selected from the group consisting of hydrogen, alkyl, cycloalkyl,alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy,aryloxy, carbonyl, acetyl, C-amido, C-thioamido, amidino, C-carboxy,O-carboxy, sulfonyl and trihalomethane-sulfonyl; R⁸ and R¹⁰ areunsubstituted lower alkyl; R⁹ is C-amido; and R^(a) and R^(b) areindependently selected from hydrogen or alkyl; or a pharmaceuticallyacceptable salt thereof.
 40. The compound of claim 39, wherein R⁹ is2-(dimethylaminoethyl) aminocarbonyl,2-(diethylaminoethyl)aminocarbonyl,2-(pyrrolidin-1-ylethyl)aminocarbonyl, or2-(morpholin-4-ylethyl)aminocarbonyl.
 41. The compound of claim 39,wherein R³ is hydrogen or lower unsubstituted alkyl; R⁴ is selected fromthe group consisting of hydrogen, halogen, aryl and S-sulfonamido; R⁵ isselected from the group consisting of hydrogen, lower alkyl, loweralkoxy, aryl, and heteroaryl; and R⁶ and R⁷ are hydrogen.
 42. Thecompound of claim 39, wherein R³ is hydrogen or methyl.
 43. The compoundof claim 39, wherein R⁴ is hydrogen, chloro, fluoro, bromo or phenyl.44. The compound of claim 42, wherein R⁴ is hydrogen or fluoro.
 45. Thecompound of claim 39, wherein R⁵ is hydrogen, methyl, ethyl, methoxy,phenyl or pyridyl.
 46. The compound of claim 45, wherein R⁵ is hydrogen.47. The compound of claim 39, wherein R^(a) and R^(b) are hydrogen. 48.A pharmaceutical composition comprising a compound of any one of claims1, 12, 15, 31 or 39 and a pharmaceutically acceptable carrier.
 49. Apharmaceutical composition comprising a compound of claim 30 and apharmaceutically acceptable carrier.
 50. The pharmaceutical compositionof claim 49, wherein said composition is administered orally.
 51. Thepharmaceutical composition of 49, wherein said composition isadministered parenterally.
 52. A method of synthesizing a compound offormula I comprising: (a) reacting a compound of the formula V:

where R³–R¹⁰ are as defined in claim 1, with an aldehyde of the formulaR^(1′)CHO, where R^(1′) is as defined in claim 1, in the presence of anorganic base, to provide a compound of formula I where R^(2′) ishydrogen; (b) optionally reacting a compound obtained in step (a) abovewith an alkylating agent, an aralkylating agent, an acylating agent or aphosphorylating agent in the presence of an organic base to provide acompound of formula I where R^(2′) is alkyl, aralkyl, aryl, acyl or—P(O)(OR)(OR′); (c) optionally removing a protecting group from theproduct of step (b); and (d) optionally forming an acid addition salt.53. A method of synthesizing a compound of formula III comprising: (a)reacting a compound of the formula V:

where R³–R¹⁰ are as defined in claim 31, with an acylating agent of theformula R^(5′)COL, where R^(5′) is as defined in claim 31 and L is aleaving group, under acylating reaction conditions, in the presence ofan organic base; (b) optionally removing a protecting group from theproduct of step (b); and (c) optionally forming an acid addition salt.54. A method of synthesizing a compound of formula IV comprising: (a)reacting a compound of the formula V:

where R³–R¹⁰ are as defined in claim 39 above, with a phosphorylatingagent of the formula XP(O)(OR^(a))(R^(b)), where R^(a) and R^(b) arealkyl and X is a leaving group under phosphorlating reaction conditionsin the presence of an organic base; (b) optionally removing the R^(a)and R^(b) groups; (c) optionally removing a protecting group from theproduct of step (b); and (d) optionally forming an acid addition or basesalt.